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Structural and functional role of leucine residues in proteins
Abstract
Circular dichroism and potentiometric titration studies of leucine random copolymers in aqueous solutions, as well as a comparison of the conformational stability in poly-α-amino acids, indicate that leucine may possibly be the amino acid with the highest propensity for forming α-helical structures. This suggests that leucine might be found most frequently in the helical regions of proteins. A survey was made on 15 different proteins containing 2473 residues with known sequence and conformation determined by X-ray crystallography: carboxy-peptidase A, α-chymotrypsin, cytochrome b5, elastase, ferricytochrome c, α- and β-hemoglobin, insulin, lysozyme, myogen, myoglobin, papain, ribonuclease A, staphylococcal nuclease, and subtilisin BPN′. It was found that 888 residues in these proteins are in helices, and 422 of them reside in the internal turns of helical regions. While Glu, Ala, Leu and His were found to be present with the highest percentages in helical regions, Leu was clearly the most abundant residue in the inner helical cores of proteins. Polar residues are found preferentially at the helix-coil boundary regions; Asp and Glu at the N-terminal and His, Lys and Arg at the C-terminal helical ends. These findings agree with Ptitsyn's (1969) analysis on seven proteins containing 1132 residues. A more comprehensive analysis in the present survey showed that Ile, Met and Val occur with the greatest frequency in the β-regions of proteins. Leu was also found as the strongest structure-forming residue in proteins (total helical and β-regions). The functional-structural role of leucine was established by showing that it occurs most frequently among residues surrounding the heme in five of the heme proteins. In addition, the greater abundance of leucine as neighbors to active-site residues in enzymes provides strong evidence that hydrophobic residues create a non-aqueous environment, aiding the polar residues in substrate binding and enzymic catalysis. Examples of conservative and non-conservative mutations of leucine in heme proteins are given to illustrate the structure—function relation of proteins, and explain why most leucine residues in the insulin, hemoglobin, and cytochrome c homologs are invariant. Finally, the strong helical-forming power of leucine, as demonstrated experimentally in synthetic copolypeptides and its high occurrence in the inner helical cores of proteins, suggests that it could have a major role as nucleation centers in the folding and evolution of large protein molecules.
... However, Leu and Met preferred β-sheet, while Ile and Val favored turn and 3 10 -helix regions, respectively (Supplementary Table 2), for sampling the alternate conformations with higher percentages. Among the 20 amino acids, Val is the highest abundance of amino acid that found in this large-scale data set (Supplementary Table 1) and known to be the most favorable structureforming amino acid in proteins (total helical and β-regions) (Chou and Fasman, 1973). This observation may be argued if this branching, fork-like hydrophobic Leu performs a functional role. ...
... This observation may be argued if this branching, fork-like hydrophobic Leu performs a functional role. Strikingly, the functional role of Leu in numerous proteins have been reported such as myoglobin, hemoglobin, ferricytochrome C, insulin etc (Chou and Fasman, 1973). Amino acids with alternate conformations in the β-sheet may be located at the edge or termini. ...
Protein dynamics linked to numerous biomolecular functions, such as ligand binding, allosteric regulation, and catalysis, must be better understood at the atomic level. Reactive atoms of key residues drive a repertoire of biomolecular functions by flipping between alternate conformations or conformational substates, seldom found in protein structures. Probing such sparsely sampled alternate conformations would provide mechanistic insight into many biological functions. We are therefore interested in evaluating the instance of amino acids adopted alternate conformations, either in backbone or side-chain atoms or in both. Accordingly, over 70000 protein structures appear to contain alternate conformations only 'A' and 'B' for any atom, particularly the instance of amino acids that adopted alternate conformations are more for Arg, Cys, Met, and Ser than others. The resulting protein structure analysis depicts that amino acids with alternate conformations are mainly found in the helical and β-regions and are often seen in high-resolution X-ray crystal structures. Furthermore, a case study on human cyclophilin A (CypA) was performed to explain the pre-existing intrinsic dynamics of catalytically critical residues from the CypA and how such intrinsic dynamics perturbed upon Ser99Thr mutation using molecular dynamics simulations on the ns-μs timescale. Simulation results demonstrated that the Ser99Thr mutation had impaired the alternate conformations or the catalytically productive micro-environment of Phe113, mimicking the experimentally observed perturbation captured by X-ray crystallography. In brief, a deeper comprehension of alternate conformations adopted by the amino acids may shed light on the interplay between protein structure, dynamics, and function.
... Repeat patterns in protein sequences have been found, although their exact role in protein structure and function has not been fully discerned (Katti et al., 2000). Experiments have shown that Leu is the strongest structure forming and neighboring active-site residue with the possible exception of Met (Chou and Fasman, 1973). Mutations from Leu to a Met residue in proteins may be considered as conservative as both residues are hydrophobic (Némethy and Scheraga, 1962), and are usually placed in highly structured interior regions of proteins near an active site (Chou and Fasman, 1973). ...
... Experiments have shown that Leu is the strongest structure forming and neighboring active-site residue with the possible exception of Met (Chou and Fasman, 1973). Mutations from Leu to a Met residue in proteins may be considered as conservative as both residues are hydrophobic (Némethy and Scheraga, 1962), and are usually placed in highly structured interior regions of proteins near an active site (Chou and Fasman, 1973). The histone family is the basic set of proteins that coordinate the organization of the eukaryotic DNA into a hierarchal structure known as chromatin (Couture and Trievel, 2006). ...
A neutral evolution model that explicitly considers codons, amino acids, and the degeneracy of the genetic code is developed. The model is built from nucleotides up to amino acids, and it represents a refinement of the neutral theory of molecular evolution. The model is based on a stochastic process that leads to a stationary probability distribution of amino acids. The latter is used as a neutral test of evolution. We provide some examples for assessing the neutrality test for a small set of protein sequences. The Jukes-Cantor model is generalized to deal with amino acids and it is compared with our neutral model, along with the empirical BLOSUM62 substitution model. The neutral test provides a baseline to which the evolution of any protein can be analyzed, and it clearly helps in discerning putative amino acids with unexpected frequencies that might be under positive or negative selection. Our model and neutral test are as universal as the standard genetic code.
... Helix, β-sheet, and coil regions The amino acid residues in the helix, β-sheet, and coil regions of these 15 proteins were tabulated in Table 2. (TABLE 2 of Chou and Fasman (1974a)). It should be noted that the β-sheet residues differ slightly from those reported earlier (Chou and Fasman, 1973) due to more recent detailed X-ray diffraction analysis references cited above. Where the β regions were not specified explicitly in the original papers, as in the case of chymotrypsin, elastase, and ribonuclease S, the schematic diagrams showing hydrogen bonding in these proteins were used to delineate the β-sheet regions. ...
... Residues at β bends which did not show hydrogen bonding were not included in the βsheet regions. All regions designated as helical (alicyll, 310, distorted helix) by the X-ray crystallographic studies have been included as helical residues in Table 2. Despite minor changes in some helical regions, based on the latest X-ray studies denoted in a following paper, the analysis did not significantly alter the calculated helix parameters so that the helical residues listed in Table 2 are identical with those reported earlier (Chou and Fasman, 1973). Ala 228 119 62 38 71 Arg 78 22 9 12 44 Asn 133 35 12 15 83 ASP 111 39 10 15 57 CYS 54 15 3 12 27 Gin 95 40 16 20 35 Glu 113 62 28 5 46 GlY 232 45 22 32 155 His 74 33 11 9 32 Ile 106 38 22 29 39 Leu 196 94 64 41 61 LYS 175 67 34 22 86 Met 28 12 6 8 8 Phe 82 33 16 18 31 Pro 85 18 0 9 58 Ser 202 57 24 25 120 Thr 156 47 21 32 77 TrP 44 18 10 9 17 Tyr 100 22 10 22 56 Val 181 74 44 51 56 Total 2473 890 424 424 1159 Before the time of Chou and Fasman, some surveys of helical and nonhelical residues had been performed, they were based on even fewer proteins (Cook, 1967;Kotelchuck et al., 1963;Ptitsyn, 1969;Finkelstein and Ptitsyn, 1971;Kabat and Wu, 1973a,b;Kabat, 1971, 1973). ...
... Helix, β-sheet, and coil regions The amino acid residues in the helix, β-sheet, and coil regions of these 15 proteins were tabulated in Table 2. (TABLE 2 of Chou and Fasman (1974a)). It should be noted that the β-sheet residues differ slightly from those reported earlier (Chou and Fasman, 1973) due to more recent detailed X-ray diffraction analysis references cited above. Where the β regions were not specified explicitly in the original papers, as in the case of chymotrypsin, elastase, and ribonuclease S, the schematic diagrams showing hydrogen bonding in these proteins were used to delineate the β-sheet regions. ...
... Residues at β bends which did not show hydrogen bonding were not included in the βsheet regions. All regions designated as helical (alicyll, 310, distorted helix) by the X-ray crystallographic studies have been included as helical residues in Table 2. Despite minor changes in some helical regions, based on the latest X-ray studies denoted in a following paper, the analysis did not significantly alter the calculated helix parameters so that the helical residues listed in Table 2 are identical with those reported earlier (Chou and Fasman, 1973). Ala 228 119 62 38 71 Arg 78 22 9 12 44 Asn 133 35 12 15 83 ASP 111 39 10 15 57 CYS 54 15 3 12 27 Gin 95 40 16 20 35 Glu 113 62 28 5 46 GlY 232 45 22 32 155 His 74 33 11 9 32 Ile 106 38 22 29 39 Leu 196 94 64 41 61 LYS 175 67 34 22 86 Met 28 12 6 8 8 Phe 82 33 16 18 31 Pro 85 18 0 9 58 Ser 202 57 24 25 120 Thr 156 47 21 32 77 TrP 44 18 10 9 17 Tyr 100 22 10 22 56 Val 181 74 44 51 56 Total 2473 890 424 424 1159 Before the time of Chou and Fasman, some surveys of helical and nonhelical residues had been performed, they were based on even fewer proteins (Cook, 1967;Kotelchuck et al., 1963;Ptitsyn, 1969;Finkelstein and Ptitsyn, 1971;Kabat and Wu, 1973a,b;Kabat, 1971, 1973). ...
... Helix, β-sheet, and coil regions The amino acid residues in the helix, β-sheet, and coil regions of these 15 proteins were tabulated in Table 2. (TABLE 2 of Chou and Fasman (1974a)). It should be noted that the β-sheet residues differ slightly from those reported earlier (Chou and Fasman, 1973) due to more recent detailed X-ray diffraction analysis references cited above. Where the β regions were not specified explicitly in the original papers, as in the case of chymotrypsin, elastase, and ribonuclease S, the schematic diagrams showing hydrogen bonding in these proteins were used to delineate the β-sheet regions. ...
... Residues at β bends which did not show hydrogen bonding were not included in the βsheet regions. All regions designated as helical (alicyll, 310, distorted helix) by the X-ray crystallographic studies have been included as helical residues in Table 2. Despite minor changes in some helical regions, based on the latest X-ray studies denoted in a following paper, the analysis did not significantly alter the calculated helix parameters so that the helical residues listed in Table 2 are identical with those reported earlier (Chou and Fasman, 1973). Ala 228 119 62 38 71 Arg 78 22 9 12 44 Asn 133 35 12 15 83 ASP 111 39 10 15 57 CYS 54 15 3 12 27 Gin 95 40 16 20 35 Glu 113 62 28 5 46 GlY 232 45 22 32 155 His 74 33 11 9 32 Ile 106 38 22 29 39 Leu 196 94 64 41 61 LYS 175 67 34 22 86 Met 28 12 6 8 8 Phe 82 33 16 18 31 Pro 85 18 0 9 58 Ser 202 57 24 25 120 Thr 156 47 21 32 77 TrP 44 18 10 9 17 Tyr 100 22 10 22 56 Val 181 74 44 51 56 Total 2473 890 424 424 1159 Before the time of Chou and Fasman, some surveys of helical and nonhelical residues had been performed, they were based on even fewer proteins (Cook, 1967;Kotelchuck et al., 1963;Ptitsyn, 1969;Finkelstein and Ptitsyn, 1971;Kabat and Wu, 1973a,b;Kabat, 1971, 1973). ...
Forty years ago, Peter Y. Chou and Gerald D. Fasman (1974a), relying on the information from fifteen proteins calculated α helix, β-sheet, and coil conformational parameters, Pα,Pβ,and Pc,for the 20 naturally occurring amino acids from the frequency of occurrence of each amino acid residue in the α, β, and coil conformations. Secondary structure of these 15 proteins had been determined by X-ray crystallography. Although the accuracy could not go over to the level of 60% too much, these values utilized for a long time to provide a simple procedure, devoid of complex computer calculations, to predict the secondary structure of proteins from their known amino acid sequences. In the same article of Peter Y. Chou and Gerald D. Fasman, a detailed analysis of the helix and β-sheet boundary residues in proteins provided amino acid frequencies at the N-and C-terminal ends which were used to delineate helical and β regions. Charged residues are found with the greatest frequency at both helical ends, but they were mostly absent in β-sheet regions. In the same article a mechanism of protein folding was proposed, whereby helix nucleation starts at the centers of the helix where the Pα values are highest, and propagates in both directions, until strong helix breakers where Pα values are lowest, terminate the growth at both ends. Similarly, residues with the highest Pβ values will initiate β regions and residues with the lowest Pβ values will terminate β regions. The helical region with the largest Pα was proposed as the site of the first fold during protein renaturation. The mechanism whereby proteins fold into their native conformation, capable of biological activity, has been a long sought after goal. With the elucidation of the three-dimensional structure of many proteins through X-ray crystallography, a new momentum has been given to understanding the factors governing this complex assembly of polypeptide chains. In this paper, using similar statistics from 20 347 proteins, the level of reliability of formerly found results is discussed.
... Branched-chain amino acids (BCAAs), such as isoleucine, leucine, and valine, are essential amino acids created by plants, bacteria, and fungi. Animals cannot synthesize BCAAs and consequently must obtain BCAAs from their diet [35,36]. BCAAs are catabolized in the mitochondria and cytoplasm of cells, where they are first converted into branched-chain α-keto acids, 2-ketoisocaproate, 2-keto-3-methylvalerate, and 2ketoisovalerate. ...
Renal cell carcinoma (RCC) is the predominant form of malignant kidney cancer. Sunitinib, a primary treatment for advanced, inoperable, recurrent, or metastatic RCC, has shown effectiveness in some patients but is increasingly limited by drug resistance. Recently identified cuproptosis, a copper-ion-dependent form of programmed cell death, holds promise in combating cancer, particularly drug-resistant types. However, its effectiveness in treating drug resistant RCC remains to be determined. Exploring cuproptosis's regulatory mechanisms could enhance RCC treatment strategies. Our analysis of data from the GEO and TCGA databases showed that the cuproptosis-related gene DBT is markedly under expressed in RCC tissues, correlating with worse prognosis and disease progression. In our study, we investigated copper CRGs in ccRCC, noting substantial expression differences, particularly in advanced-stage tumors. We established a connection between CRG expression levels and patient survival, positioning CRGs as potential therapeutic targets for ccRCC. In drug resistant RCC cases, we found distinct expression patterns for DBT and GLS CRGs, linked to treatment resistance. Our experiments demonstrated that increasing DBT expression significantly reduces RCC cell growth and spread, underscoring its potential as a therapeutic target. This research sheds new light on the role of CRGs in ccRCC and their impact on drug resistance.
... Among Glu, Ala, Leu, and His, which exhibit the highest occurrences within helical regions, Leu stands out as the predominant residue within the central helical cores of proteins. Therefore a high frequency of Leu suggests that these proteins may have a stable core structure [50]. From the above observations, we can conclude that such a high frequency of amino acids being at the maximal and minimal ends is suggestive of a probable enormous functional diversity. ...
Moonlighting proteins, known for their ability to perform multiple, often unrelated functions within a single polypeptide chain, challenge the traditional "one gene, one protein, one function" paradigm. As organisms evolved, their genomes remained relatively stable in size, but the introduction of post-translational modifications and sub-strategies like protein promiscuity and intrinsic disorder enabled multifunctionality. Enzymes, in particular, exemplify this phenomenon, engaging in unrelated processes alongside their primary catalytic roles. This study employs a systematic, quantitative informatics approach to shed light on human moonlighting protein sequences. Phylogenetic analyses of human moonlighting proteins are presented, elucidating the distal-proximal relationships among these proteins based on sequence-derived quantitative features. The findings unveil the captivating world of human moonlighting proteins, urging further investigations in the emerging field of moonlighting proteomics, with the potential for significant contributions to our understanding of multifunctional proteins and their roles in diverse cellular processes and diseases.
... The α-helical content of mutant mAcP upon TFE-denaturation is higher than the WT, meaning that substitutions to leucine favour a rapid structural transition into non-native αhelices. This observation is in agreement with the fact that leucine has a higher α-helix propensity than threonine, serine or glutamine, and the highest frequency in the α-helical strands of globular proteins, with stabilising effects towards α-helical folding (57,68). The presence of osmolytes further enhances the amount of α-helical structureat time zero of mAcP aggregation kinetics, as osmolytes provide an additive contribution to the stability of the newly formed helices (8,30,69). ...
... It is important to mention that the non-polar aliphatic amino acids are important in the formation of a non-aqueous environment, aiding the polar residues in substrate binding and enzymatic catalysis. 43,44 However, crystallographic studies are required to corroborate the bioinformatic information and to be able to identify the amino acid residues that are critical for the binding of the 3H-indole-3-one. We tried to predict the type of photoreceptor based on its possible subcellular location. ...
Cap color is an important commercial trait for oyster mushrooms. Various pigment constituents determine a diverse color. However, the pigments of oyster mushrooms are still ambiguous. The pink oyster mushroom (Pleurotus salmoneostramineus or Pleurotus djamor) chromoprotein is one of the few proteins belonging to this fungus that has a record of its sequence of amino acid residues. However, even though there are studies about this chromoprotein isolation, purification, and crystallization, the current information focused on its 3-dimensional model and the cofactor and prosthetic group (3H-indol-3-one) binding sites is unreliable and fragmented. Therefore, in this study, using free online servers such as Prot pi, GalaxyWEB, MIB, and CB-Dock2, a structural analysis and the prediction of its physicochemical and biological properties were conducted, to understand the possible function of this chromoprotein. The obtained results showed that this molecule is a protein with a molecular weight of 23 712.5 Da, an isoelectric point of 7.505, with oligomerization capacity in a dimer and glycation in the Ser6 residue. In addition, the participation of the residues Leu5, Leu8, Lys211, Ala214, and Gln215 in the binding of the prosthetic group to the protein was highlighted; as well as Ser6 and Pro7 are important residues for the interaction of the Mg2+ ion and eumelanin. Likewise, morphological changes based on different culture conditions (light/dark) showed that this protein is constitutive expressed and independent of blue light. The findings in this study demonstrate that pink chromoprotein is a melanosomal protein, and it possibly has a critical role in melanogenesis and the melanin polymerization. However, more experimental studies are needed to predict a possible mechanism of action and type of enzymatic activity.
... The basic side chains of these aa's are known to form extensive H-bonds with the DNA-phosphate backbone to strengthen the protein-DNA interactions (Wang et al. 2020). Also, the presence of leucine at specific intervals in the inner core of the α-helices of MYB DBD of groundnut creates more tendency than other amino acids for building the α-helical structures (Chou and Fasman 1973). Apart from the conserved pattern of Trp and Leu, there were some random conserved aa's that were also found in MYB DBD of groundnut, which helps in the normal functioning of the MYB proteins (Wang et al. 2020). ...
The myeloblastosis (MYB) superfamily is one of the largest transcription factor families in the plant kingdom, and has diverse functions in plants, including regulating biotic and abiotic stresses. In this study, a total of 108 MYB protein sequences were identified and characterized through an insilico approach. We confirmed that the MYB proteins had three conserved repeats (R1, R2, and R3). The putative functions of AhyMYB genes were predicted on the basis of 31 functional groups formed from a comparative phylogenetic analysis. Important cisregulatory motifs, such as ABRE, TGA, ARE, LTR and TATA were present in the upstream regions of AhyMYB genes, and also some post-translational modifications were identified in the present study. Drought- stress was induced in five elite groundnut genotypes (Dh 256, Dh 257, GPBD 4, TMV 2, and JL 24) at flowering and peg initiation stages, and the expression patterns of five selected AhyMYB genes were investigated in these genotypes. In the drought-tolerant genotypes (Dh 256 and Dh 257), at the peg initiation stage, five drought-related transcription MYB factor genes showed greater up-regulation compared with the drought-susceptible genotypes. Among the five genes used in the study, AhyMYB17 gene showed higher expression in JL 24 (moderately drought resistant) compared to untreated control plants at the peg initiation stage. These results indicated the possible involvement of MYB transcription factor genes in regulating the drought stress conditions in groundnut. In-silico analysis, along with the expression studies of AhyMYB genes will definitely help in understanding the stress-response mechanism in groundnut.
... Branched chain amino acids (BCAAs), valine (Val), leucine (Leu) and isoleucine (Ile), are essential amino acids that cannot be synthesized by animals, but only from bacteria, plants and fungi and must be obtained from diet sources. These amino acids have branched hydrophobic side chains and play a primary role in protein structure [1]. All three BCAAs account for approximately 20-25% of most dietary proteins and constitute about 35% of the essential amino acids in mammals [2]. ...
Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications.
... Several inhibitors are known for enzymes such as AHAS, IlvC and DHAD belonging to BCAA biosynthesis [9][10][11][12][13][14][15]. BCAAs have a role in determining the secondary structures of proteins, with leucine (Leu) being involved in the stabilization of the α-helical structure of proteins and present in the inner helical core of proteins, while isoleucine (Ile) and valine (Val) are preferentially present in β-sheets [16,17]. BCAAs also play a role in signalling, physiological adaptations against stresses and in virulence and survival [18]. ...
... Meat, fish, egg, and dairy products have a high content of BCAAs. Similar to other amino acids, BCAAs play an important role in protein synthesis and neurotransmitter synthesis [12][13][14][15][16]. In addition, BCAAs also modulate food intake and glycemic control via influencing hormones release, such as leptin, glucagon-like peptide-1, and ghrelin [17][18][19] (see [20,21] for a general review of BCAA metabolism). ...
Branched-chain amino acids (BCAAs) are essential amino acids which have critical roles in protein synthesis and energy metabolism in the body. In the heart, there is a strong correlation between impaired BCAA oxidation and contractile dysfunction in heart failure. Plasma and myocardial levels of BCAA and their metabolites, namely branched-chain keto acids (BCKAs), are also linked to cardiac insulin resistance and worsening adverse remodelling in the failing heart. This review discusses the regulation of BCAA metabolism in the heart and the impact of depressed cardiac BCAA oxidation on cardiac energy metabolism, function, and structure in heart failure. While impaired BCAA oxidation in the failing heart causes the accumulation of BCAA and BCKA in the myocardium, recent evidence suggested that the BCAAs and BCKAs have divergent effects on the insulin signalling pathway and the mammalian target of the rapamycin (mTOR) signalling pathway. Dietary and pharmacological interventions that enhance cardiac BCAA oxidation and limit the accumulation of cardiac BCAAs and BCKAs have been shown to have cardioprotective effects in the setting of ischemic heart disease and heart failure. Thus, targeting cardiac BCAA oxidation may be a promising therapeutic approach for heart failure.
... The functional R groups of all three BCAAs are branched (hence their name), small, and hydrophobic, rendering them critical components of most proteins (Chou and Fasman 1973;Dill 1990 ...
The global prevalence of obesity and type 2 diabetes (T2D) continues to rise at an alarming rate. Despite the well-established association between obesity and an increased risk of developing T2D, the mechanisms underlying the pathogenesis of T2D with obesity remain unclear. Skeletal muscle is a major site for the disposal of ingested carbohydrate in healthy individuals. It is generally accepted that chronic overnutrition leads to accumulation of fat and fatty acid metabolites within the skeletal muscle which are believed to play a pivotal role in the progression of insulin resistance to carbohydrate metabolism which is one of the pathological hallmarks of T2D. Whilst several metabolites have been implicated, there appears to be no consensus over which metabolite is mediating skeletal muscle insulin resistance. In recent years, circulating concentrations of acylcarnitines, which are intermediates in glucose, fatty acid and branched chain amino acids (BCAAs) metabolism have been identified as potential novel biomarkers of insulin resistance and T2D. Furthermore, fatty acid derived acylcarnitines have since been shown to impair insulin signalling in vitro. Interestingly, BCAAs and their associated short-chain acylcarnitines appear to be more closely associated with insulin resistance than any marker of fatty acid metabolism, giving rise to the hypothesis that BCAAs and their catabolites may also play a causative role in the development of skeletal muscle insulin resistance. However, the simultaneous and quantitative assessment of acylcarnitines, BCAAs and related metabolites in human skeletal muscle is lacking or limited to one metabolite group. Therefore, the main aim of this this thesis was to develop a quantitative analytical method for the assessment of BCAAs and acylcarnitines in human muscle to extend upon much of published literature which has been limited to investigations in fasting plasma samples and to explore their role in the development of insulin resistance and T2D. In Chapter 3 a novel liquid chromatography coupled to high resolution mass spectrometry method was developed and optimised to enable the quantitative assessment of a full range of BCAA and fatty acid derived acylcarnitines, BCAAs and related catabolites in both human plasma and muscle samples. The commonly cited challenges of metabolite quantification from biological tissues were systematically addressed using stable isotope internal standards allowing metabolite concentrations to be determined with a high degree of confidence. In Chapter 4, the method was validated by quantitatively assessing BCAAs and acylcarnitine concentrations in fasting plasma and skeletal muscle samples of patients with T2D and an age matched obese control subjects. The results revealed striking elevations of BCAAs and BCAA derived acylcarnitines in both plasma and muscle of patients with T2D compared to control subjects. Furthermore, these metabolites were significantly correlated with fasting blood glucose. Surprisingly, no significant differences in fatty acid derived acylcarnitines were observed between groups in either plasma or muscle. These data show that plasma profiles may not always reflect muscle profiles as suggested by previous reports. In Chapter 5, skeletal muscle BCAA and acylcarnitine metabolism was investigated in the fasted and insulin stimulated state. In order to determine if the elevations observed in fasting state in the previous persist in the face of insulin thereby allowing some indication of whether they could be causative of insulin resistance. A group of young and old lean, and old overweight/obese individuals was investigated in a cross sectional design. Ageing was associated with a (30%) decline in muscle BCAA content and decreased insulin sensitivity. And increased adiposity was associated with a (20%) increase in BCAA content. In response to insulin infusion, there was an attenuated decline in muscle BCAA and BCAA catabolite content in the old lean group only. In addition, fatty acid derived acylcarnitines were suppressed in all groups despite differing glucose disposal during insulin infusion. These findings appear to dissociate muscle BCAA content, ageing and insulin resistance and suggest fatty acid derived acylcarnitnes may not be associated with insulin resistance. In Chapter 6, the potential interactions of BCAA and fatty acid metabolism were explored in a group of middle aged and older aged patients with T2D. Middle aged T2D patients had elevated meal derived fatty acid oxidation and endogenous fatty acid delivery to muscle during an oral glucose tolerance test (OGTT) when compared to age and BMI matched control subjects. This was associated with elevated fasting muscle BCAA content and an attenuated suppression of plasma BCAA and BCAA catabolites during the OGTT. Remarkably, these metabolic perturbations were absent in the older aged T2D patients, despite a similar duration of diabetes and insulinaemic responses to OGTT. Collectively, the work in this thesis provides quantitative assessment of muscle BCAA catabolism and fatty acid metabolism in humans. T2D and obesity-induced insulin resistance are characterised by elevated BCAAs and BCAA derived acylcarnitines but ageing per se exerts the opposite effects. In addition, the results of this thesis suggest that fatty acid derived acylcarnitines may not be associated with muscle insulin resistance in vivo. The potential role of BCAA derived acylcarnitines in insulin resistance and their role as biomarkers of progression to T2D requires further investigation.
... Despite this, there are slight differences between them. Leucine is a common component of alpha helices (33). However, valine and isoleucine possess additional methyl groups that cause steric hindrance, thus relegating them to beta-sheet structures (34). ...
Anthrax disease is caused by infection with the bacteria Bacillus anthracis which, if left untreated, can result in fatal bacteremia and toxemia. Current treatment for infection requires prolonged administration of antibiotics. Despite this, inhalational and gastrointestinal anthrax still result in lethal disease. By identifying key metabolic steps that B. anthracis uses to grow in host-like environments, new targets for antibacterial strategies can be identified. Here, we report that the ilvD gene, which encodes dihydroxyacid dehydratase in the putative pathway for synthesizing branched chain amino acids, is necessary for B. anthracis to synthesize isoleucine de novo in an otherwise limiting microenvironment. We observed that Δ ilvD B. anthracis cannot grow in media lacking isoleucine, but growth is restored when exogenous isoleucine is added. In addition, ΔilvD bacilli are unable to utilize human hemoglobin or serum albumin to overcome isoleucine auxotrophy, but can when provided with the murine forms. This species-specific effect is due to the lack of isoleucine in human hemoglobin. Furthermore, even when supplemented with physiological levels of human serum albumin, apotransferrin, fibrinogen, and IgG, the ilvD knockout strain grew poorly relative to non-supplemented wild-type. In addition, comparisons upon infecting humanized mice suggest that murine hemoglobin is a key source of isoleucine for both WT and Δ ilvD bacilli. Further growth comparisons in murine and human blood show that the auxotrophy is detrimental for growth in human blood, not murine. This report identifies ilvD as necessary for isoleucine production in B. anthracis , and that it plays a key role in allowing the bacilli to effectively grow in isoleucine poor hosts.
Importance
Anthrax disease, caused by B. anthracis , can cause lethal bacteremia and toxemia, even following treatment with antibiotics. This report identifies the ilvD gene, which encodes a dihydroxyacid dehydratase, as necessary for B. anthracis to synthesize the amino acid isoleucine in a nutrient-limiting environment, such as its mammalian host. The use of this strain further demonstrated a unique species-dependent utilization of hemoglobin as an exogenous source of extracellular isoleucine. By identifying mechanisms that B. anthracis uses to grow in host-like environments, new targets for therapeutic intervention are revealed.
... The results of KEGG showed that its target genes were mostly enriched in valine, leucine, and isoleucine degradation pathway. Valine, leucine, and isoleucine all have functional R group branches, therefore, they are referred to as branched chain amino acids (BCAA) [44]. They are important nutrients and metabolic regulators that are beneficial to health. ...
Objective: The pancreatic endocrinal system dominates the regulation of blood glucose levels in vivo, and the dysfunction of pancreatic endocrine β-cells is a major cause of the occurrence and development of Type 2 diabetes (T2D). Although microRNA (miRNA) have been found to be key regulators of pancreatic β-cells proliferation, differentiation and apoptosis, the underlying mechanism remains enigmatic. The aim of this study was to identify several novel miRNAs which might be involved in the etiopathogenesis of diabetic β-cells dysfunction.
Methods: The miRNA expression profiles in the pancreas of high-fat diet (HFD) fed Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats feed with normal-fat diet (NFD) were detected by using miRNA microarray chip, and individually verified the most significant factors by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to predict the target genes related to each of the identified miRNAs and the functions of these target genes in different metabolic signaling pathways.
Results: Compared with the ZL rats, a total of 24 differentially expressed miRNAs were detected in ZDF rats. Among which miR-34a-5p and miR-452-5p were the most significantly up-regulated and down-regulated respectively. These miRNAs have not been reported in rats' pancreas before. By GO and KEGG enrichment analyses, we found that miR-34a-5p could negatively regulate pancreatic β-cell proliferation through the involvement of Wnt signaling pathway. In addition, it was also found to regulate insulin secretion through the insulin signaling pathway to modulate blood glucose levels. At the same time, miR-452-5p was found to positively regulate the activity of the key rate-limiting enzyme branched-chain α-keto acid dehydrogenase-β (BCKDHB) in the catabolism of branched chain amino acids (BCAA), leading to mitochondrial dysfunction in pancreatic β-cells.
Conclusions: miR-34a-5p and miR-452-5p were identified as the novel regulators of pancreatic endocrine dysfunction. These miRNAs might have the potential to be utilized as the new predictive biomarkers for the diagnosis of the occurrence and development of T2D, as well as the therapeutic targets for T2D treatment.
... The presence of high leucine content in ORF7a protein, ORF7b protein, E-protein, surface glycoprotein, ORF1ab polyprotein, ORF1a polyprotein, M-protein, ORF6 protein, ORF3a protein and ORF8 protein is the indication of their important roles in various protein-protein interaction processes (Supplementary Figure 3). The strong helicalforming power of leucine, as demonstrated experimentally in synthetic co-polypeptides and its high occurrence in the inner helical cores of proteins, suggests that it could have a major role as nucleation centers in the folding and evolution of large protein molecules [33]. ...
This study reports sequence data mining and analysis, complete coordinate tertiary structure prediction including Deep Learning
inspired validation, and in silico functional characterization of the full SARS-CoV-2 proteome based on the NCBI reference sequence
NC_045512 (29903 bp ss-RNA). Out of 25 polypeptides analyzed, 3D structures of 15 of them were predicted using comparative
protein structure prediction method and ab-initio modelling method due to unavailability of experimentally determined structures.
Deep Learning and Neural Network based tools such as QMEANDisCo 4.0.0, MolProbity 4.4, ProQ3D and Procheck were used to
verify the predicted 3D structures. Tunnel analysis revealed the presence of multiple tunnels in NSP4, nucleocapsid phosphoprotein,
NSP3, membrane glycoprotein, ORF6 protein, NSP1, NSP6, and envelop protein, indicating a large number of transport pathways
for small ligands that influence their reactivity. Ligand-binding pockets with high estimates of druggability scores were detected in
envelope glycoprotein (0.97), membrane glycoprotein (0.87), NSP6 (0.79), ORF7a (0.79), ORF8 (0.75), ORF3a (0.72), and NSP4
(0.70), indicating the ability to bind drug-like molecules with high affinity indicating that the predicted structures would be useful
for protein nanotechnology in understanding protein machinery towards drug repurposing and discovery studies. Moreover, the
molecular phylogenetic analysis of orf1ab polyprotein indicates close relatedness of SARS-CoV-2 to the bat coronavirus.
... Although first discovered and studied extensively since the late 1800s, it was the last 20 years or so during which branchedchain amino acids (BCAAs) have emerged as vital elements in various aspects of health and diseases as investigators began to unfold their roles beyond protein synthesis and degradation. BCAAs comprise leucine, isoleucine, and valine that have branched functional R groups [1]. Known as the essential amino acids due to the inability of animals to synthesize them (unlike microbial organisms and plants), BCAAs must be obtained through diet sources. ...
Branched-chain amino acids (BCAAs) are essential amino acids that are not synthesized in our body; thus, they need to be obtained from food. They have shown to provide many physiological and metabolic benefits such as stimulation of pancreatic insulin secretion, milk production, adipogenesis, and enhanced immune function, among others, mainly mediated by mammalian target of rapamycin (mTOR) signaling pathway. After identified as a reliable marker of obesity and type 2 diabetes in recent years, an increasing number of studies have surfaced implicating BCAAs in the pathophysiology of other diseases such as cancers, cardiovascular diseases, and even neurodegenerative disorders like Alzheimer's disease. Here we discuss the most recent progress and review studies highlighting both correlational and potentially causative role of BCAAs in the development of these disorders. Although we are just beginning to understand the intricate relationships between BCAAs and some of the most prevalent chronic diseases, current findings raise a possibility that they are linked by a similar putative mechanism.
... globular proteins) as well as the interaction of the transmembrane domains of membranous proteins with phospholipid bilayers (Brosnan and Brosnan, 2006;Dill, 1990). This characteristic of BACCs was also important for substrate binding and catalysis of various enzymes (Chou and Fasman, 1973). Thus the downregulation of genes related to BCAA degradation in K. mikimotoi may be undertaken to preserve membrane integrity, to prevent protein degradation and to maintain the stability and maturation of newly synthesized protein (Poong et al., 2017). ...
Solar ultraviolet radiation (UVR) is a stress factor in aquatic environments and may act directly or indirectly on orgnisms in the upper layers of the water column. However, UVR effects are usually species-specific and difficult to extrapolate. Here we use the HAB-forming, toxic dinoflagellate Karenia mikimotoi (which was found to be
relatively resistant in previous studies) to investigate its transcriptional responses to a one-week UVR exposure. For this, batch cultures of K. mikimotoi were grown with and without UVR, and their transcriptomes (generated via RNAseq technology) were compared. RNA-seq generated 45.31 million reads, which were further assembled
to 202600 unigenes (> 300bp). Among these, ca. 61% were annotated with NCBI, NR, GO, KOG, PFAM, Swiss-Prot, and KEGG database. Transcriptomic analysis revealed 722 differentially expressed unigenes (DEGs, defined as being within a |log2 fold change| ≥ 2 and padj < 0.05) responding to solar UVR, which were only 0.36% of
all unigenes. 716 unigenes were down-regulated, and only 6 unigenes were up-regulated in the UVR compared to non-UVR treatment. KEGG pathway further analysis revealed DEGs were involved in the different pathway; genes involved in the ribosome, endocytosis and steroid biosynthesis pathways were highly down-regulated, but this was not the case for those involved in the energy metabolisms (including photosynthesis, oxidative phosphorylation) which may contribute to the sustainable growth observed in UVR treatment. The up-regulated
expression of both zinc-finger proteins (ZFPs) and ribosomal protein L11 (RPL11) may be one of the acclimated mechanisms against UVR. In addition, this work identified down-regulated genes involved in fatty acid degradation and the hydrophobic branched chain amino acids (e.g., Valine, leucine, and isoleucine), which act as structural components of cell membranes modulating lipid homeostasis or turnover. In conclusion, the present study suggests that the toxic dinoflagellate K. mikimotoi has limited transcriptomic regulation but confirms that it appears as a tolerant species in response to solar UVR. These findings expand current knowledge of gene expression in HAB-forming species in response to natural environment factors such as solar radiation.
... The BCAAs are small nonpolar amino acids with branched alkyl side chains that make them hydrophobic and confer unique properties in proteins. Leu is a strong stabilizer of ␣-helical structures and, as such, is typically found in the inner helical core of proteins (7), whereas the substitution of the -carbon with a methyl group on Ile and Val creates bulkiness that destabilizes ␣-helical structures; thus, Ile and Val are preferentially located in -sheets (8,9). ...
The branched-chain amino acids (BCAAs [Ile, Leu, and Val]) represent important nutrients in bacterial physiology, with roles that range from supporting protein synthesis to signaling and fine-tuning the adaptation to amino acid starvation. In some pathogenic bacteria, the adaptation to amino acid starvation includes induction of virulence gene expression: thus, BCAAs support not only proliferation during infection, but also the evasion of host defenses. A body of research has accumulated over the years to describe the multifaceted physiological roles of BCAAs and the mechanisms bacteria use to maintain their intracellular levels. More recent studies have focused on understanding how fluctuations in their intracellular levels impact global regulatory pathways that coordinate the adaptation to nutrient limitation, especially in pathogenic bacteria. In this minireview, we discuss how these studies have refined the individual roles of BCAAs, shed light on how BCAA auxotrophy might promote higher sensitivity to exogenous BCAA levels, and revealed pathogen-specific responses to BCAA deprivation. These advancements improve our understanding of how bacteria meet their nutritional requirements for growth while simultaneously remaining responsive to changes in environmental nutrient availability to promote their survival in a range of environments.
... Leucine is in fact the most abundant sidechain in all proteins [321] and its abundance has been linked to its ability to adopt multiple conformations [322]. In addition to its higher relative abundance, leucine rich regions commonly have specific roles as nuclear export signals [323,324], in DNA binding [325] or as neighbors to active sites [326]. Furthermore, transmembrane helices are enriched in leucine [327] and oligoleucines, but not oligoalanines, were shown to self-assemble in membranes [328]. ...
In the last 20 years, an increasing number of studies have been reported on membrane active peptides. These peptides exert their biological activity by interacting with the cell membrane, either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. Membrane active peptides are attractive alternatives to currently used pharmaceuticals and the number of antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery in the drug pipeline is increasing. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). Antimicrobial peptides are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus, and fungi. Cell penetrating peptides are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity. Biophysical techniques shed light on peptide–membrane interactions at higher resolution due to the advances in optics, image processing, and computational resources. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Live imaging techniques allow examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provide clues on the peptide–lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.
... Furthermore, the percentage richness of amino acid showed the presence of leucine and glutamic acid-rich profile for insect trehalases (S6). It has been observed that amino acids, namely, Glu, Ala, Leu and His occur in high percentages in the helical regions of a protein with Leu being the most abundant residue present in the inner helical cores of α helix-rich proteins (Chou and Fasman 1973;Kumar and Bansal 1998). ...
Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57–92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71–81). The modelled 3D tertiary structure of CrTre depicts a (α/α)6 barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.
... Leucine is in fact the most abundant sidechain in all proteins [303] and its abundance has been linked to its ability to adopt multiple conformations [304]. In addition to its higher relative abundance, leucine rich regions commonly have specific roles as nuclear export signals [305,306], in DNA binding [307] or as neighbors to active sites [308]. Furthermore, transmembrane helices are enriched in leucine [309] and oligoleucines, but not oligoalanines, were shown to selfassemble in membranes [310]. ...
In the last 20 years, an increasing number of studies have been reported on membrane active peptides, which exert their biological activity by interacting with the cell membrane either to disrupt it and lead to cell lysis or to translocate through it to deliver cargos into the cell and reach their target. These peptides are attractive alternatives to currently used pharmaceuticals. Antimicrobial peptides (AMPs) and peptides designed for drug and gene delivery currently in the drug pipeline suggest that these membrane active peptides will soon constitute a significant percentage of the drug market. Here, we focus on two most prominent classes of membrane active peptides; AMPs and cell-penetrating peptides (CPPs). AMPs are a group of membrane active peptides that disrupt the membrane integrity or inhibit the cellular functions of bacteria, virus and fungi. CPPs are another group of membrane active peptides that mainly function as cargo-carriers even though they may also show antimicrobial activity to some extent. Biophysical techniques to understand how they interact with the membrane have shed light on the peptide–membrane interaction at various levels of detail. Structural investigation of membrane active peptides in the presence of the membrane provides important clues on the effect of the membrane environment on peptide conformations. Advances in live imaging techniques have allowed examination of peptide action at a single cell or single molecule level. In addition to these experimental biophysical techniques, molecular dynamics simulations provided clues on the peptide-lipid interactions and dynamics of the cell entry process at atomic detail. In this review, we summarize the recent advances in experimental and computational investigation of membrane active peptides with particular emphasis on two amphipathic membrane active peptides, the AMP melittin and the CPP pVEC.
... Branched-chain amino acids (BCAA) are the most hydrophobic amino acids and play an important role in formation of transmembrane proteins. Furthermore, BCAA make up 37% of the amino acids in lung surfactant protein B (Chou and Fasman, 1973). The changes observed for BCAA (leucine 1.5 fold and isoleucine 1.4-fold) at 24 h suggest an ongoing repair process of lipid membranes and surfactant in the airway. ...
Controlled human exposure to the oxidant air pollutant ozone causes decrements in lung function and increased inflammation as evidenced by neutrophil influx into the lung and increased levels of proinflammatory cytokines in the airways. Here we describe a targeted metabolomics evaluation of human bronchioalveolar lavage fluid (BALF) following controlled in vivo exposure to ozone to gain greater insight into its pulmonary effects. In a two-arm cross-over study, each healthy adult human volunteer was randomly exposed to filtered air (FA) and to 0.3 ppm ozone for 2 hr while undergoing intermittent exercise with a minimum of 4 weeks between exposures. Bronchoscopy was performed and BALF obtained at 1 (n = 9) or 24 (n = 23) h post-exposure. Metabolites were detected using ultrahigh performance liquid chromatography-tandem mass spectroscopy. At 1-hour post-exposure, a total of 28 metabolites were differentially expressed (DE) (p < 0.05) following ozone exposure compared to FA-exposure. These changes were associated with increased glycolysis and antioxidant responses, suggesting a rapid increased energy utilization as part of the cellular response to oxidative stress. At 24-hour post-exposure, 41 metabolites were DE. Many of the changes were in amino acids and linked with enhanced proteolysis. Changes associated with increased lipid membrane turnover were also observed. These later-stage changes were consistent with ongoing repair of airway tissues. There were 1.37 times as many metabolites were differentially expressed at 24 hour compared to 1-hour post-exposure. The changes at 1 hour reflect responses to oxidative stress while the changes at 24 hour indicate a broader set of responses consistent with tissue repair. These results illustrate the ability of metabolomic analysis to identify mechanistic features of ozone toxicity and aspects of the subsequent tissue response.
... The hydrophobicity of these BCAAs is important for their role in globular proteins, membranous proteins and coiled-coil structures not only for the stability of the folded protein but also for the folding pathway leading to the mature structure (Dill 1990). This characteristic is also important for substrate binding and catalysis of various enzymes (Chou and Fasman 1973). BCAAs inhibit protein degradation and enhance protein synthesis through the formation of aminoacyl-tRNA derivatives (Freund and Hanani 2002). ...
Stratospheric ozone depletion has led to increasing levels of ultraviolet radiation (UVR) reaching the Earth’s surface. Elevated UVR, particularly in the high latitudes, potentially causes shifts in species composition and diversity in various ecosystems, consequently altering the biogeochemical cycles. Microalgae are not only ecologically important as primary producers, generating atmospheric oxygen and sequestering carbon dioxide; they are also economically important as sources of health supplement, pigments, biofuel and others. Changes to the size and composition of algal communities can lead to profound impacts to the fisheries productivity. There have been studies on the effects of UVR on the growth, photosynthesis and biochemical composition of microalgae, but limited information on the underlying molecular mechanisms involved in the response and adaptation of microalgae to UVR is available. We employed RNA-seq to quantitatively evaluate and compare the transcriptomes of an Antarctic freshwater Chlorella sp. grown at ambient versus elevated UVR conditions. Differentially expressed genes, relating to the fatty acid degradation, amino acid metabolism, starch and sucrose metabolism and peroxisome pathways, suggest conservation and remobilisation of energy resources, maintenance of newly synthesised protein and inhibition of protein degradation, ensuring membrane lipid homeostasis and regulating antioxidative mechanisms, as the acclimation strategies in response to UVR. These findings expand current knowledge of gene expression in polar Chlorella sp. in response to short-term UVR. Studies on stress tolerance mechanisms are important to understand and predict future impacts of climate change. Genes, proteins and pathways identified from these adaptable polar algae have potentially far-reaching biotechnological applications.
... BCAAs form the helical zipper structures of transcription factors ( Chou and Fasman, 1978;Glover and Harrison, 1995). Moreover, BCAAs are present in most of the non-aqueous interior environment of water-soluble globular proteins, such as the oxygen-binding portion of myoglobin and haemoglobin ( Chou and Fasman, 1973). ...
Substantial evidence has been accumulated suggesting that branched-chain amino acid (BCAA) supplementation or BCAA-rich diets have a positive effect on the regulation of body weight, muscle protein synthesis, glucose homeostasis, the ageing process and extend healthspan. Despite these beneficial effects, epidemiological studies have shown that BCAA plasma concentrations and BCAA metabolism are altered in several metabolic disorders, including type 2 diabetes mellitus and cardiovascular diseases. In this review article, we present an overview of the current literature on the different effects of BCAAs in health and disease. We also highlight the results showing the most promising therapeutic effects of dietary BCAA supplementation and discuss how BCAAs can trigger different and even opposite effects, depending on the catabolic and anabolic states of the organisms. Moreover, we consider the effects of BCAAs when metabolism is abnormal, in the presence of a mixture of different anabolic and catabolic signals. These unique pharmacodynamic properties may partially explain some of the markedly different effects found in BCAA supplementation studies. To predict accurately these effects, the overall catabolic/anabolic status of patients should be carefully considered. In wider terms, a correct modulation of metabolic disorders would make nutraceutical interventions with BCAAs more effective.
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This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.
... This antiparallel orientation is in accordance with an assumed head-to-tail configuration of the channels [10]. The fact that leucine to alanine mutations in the inner helix drastically impaired channel function is in accordance with the observation that although alanine is a residue of high helix propensity, it can decrease the stability of the helix (dimer) [66,68]. Indeed, coarse-grained MD simulations discovered mutated dimers that exhibit a significant increase of flexibility, pointing to less stable dimers. ...
Two-pore channels (TPCs) constitute a family of intracellular cation channels with diverse permeation properties and functions in animals and plants. In the model plant Arabidopsis, the vacuolar cation channel TPC1 is involved in propagation of calcium waves and in cation homeostasis. Here, we discovered that the dimerization of a predicted helix within the carboxyl-terminus (CTH) is essential for the activity of TPC1. Bimolecular fluorescence complementation and co-immunoprecipitation demonstrated the interaction of the two C-termini and pointed towards the involvement of the CTH in this process. Synthetic CTH peptides dimerized with a dissociation constant of 3.9 µM. Disruption of this domain in TPC1 either by deletion or point mutations impeded the dimerization and cation transport. The homo-dimerization of the CTH was analyzed in silico using coarse-grained molecular dynamics (MD) simulations for the study of aggregation, followed by atomistic MD simulations. The simulations revealed that the helical region of the wild type, but not a mutated CTH forms a highly stable, antiparallel dimer with characteristics of a coiled-coil. We propose that the voltage- and Ca²⁺-sensitive conformation of TPC1 depends on C-terminal dimerization, adding an additional layer to the complex regulation of two-pore cation channels.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-016-2131-3) contains supplementary material, which is available to authorized users.
... Based on these data it is reasonable to predict that milk-derived microRNA-29b also targets the mRNA of dihydrolipoyl branched-chain acyltransferase, disintegrating the BCKD metabolon of the milk consumer. This potential postnatal inhibition of BCAA catabolism during the lactation period may rescue valuable BCAAs from mitochondrial oxidation, thereby raising the BCAA serum levels required for mTORC1-dependent translation as well as synthesis of functionally and structurally BCAA-rich proteins such as hemoglobin, myoglobin, multiple enzymes, leucine zippers, surfactant protein B, and other BCAA-enriched proteins [126][127][128][129]. ...
Based on own translational research of the biochemical and hormonal effects of cow's milk consumption in humans, this review presents milk as a signaling system of mammalian evolution that activates the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the pivotal regulator of translation. Milk, a mammary gland-derived secretory product, is required for species-specific gene-nutrient interactions that promote appropriate growth and development of the newborn mammal. This signaling system is highly conserved and tightly controlled by the lactation genome. Milk is sufficient to activate mTORC1, the crucial regulator of protein, lipid, and nucleotide synthesis orchestrating anabolism, cell growth and proliferation. To fulfill its mTORC1-activating function, milk delivers four key metabolic messengers: (1) essential branched-chain amino acids (BCAAs); (2) glutamine; (3) palmitic acid; and (4) bioactive exosomal microRNAs, which in a synergistical fashion promote mTORC1-dependent translation. In all mammals except Neolithic humans, postnatal activation of mTORC1 by milk intake is restricted to the postnatal lactation period. It is of critical concern that persistent hyperactivation of mTORC1 is associated with aging and the development of age-related disorders such as obesity, type 2 diabetes mellitus, cancer, and neurodegenerative diseases. Persistent mTORC1 activation promotes endoplasmic reticulum (ER) stress and drives an aimless quasi-program, which promotes aging and age-related diseases.
... Leucine, isoleucine and valine are among the most hydrophobic amino acids, which are crucial determinants for hydrophobic clefts of enzymes and are important for the insertion of proteins such as receptors into cell membranes [143]. Hydrophobic residues are crucial for oxygen binding in hemoglobin and myoglobin and substrate binding of various enzymes [148]. Remarkably, surfactant protein B contains 37% of BCAAs (17.7% leucine) [149]. ...
Milk, the secretory product of the lactation genome, promotes growth of the newborn mammal. Milk delivers insulinotropic amino acids, thus maintains a molecular crosstalk with the pancreatic β-cell of the milk recipient. Homeostasis of β-cells and insulin production depend on the appropriate magnitude of mTORC1 signaling. mTORC1 is activated by branched-chain amino acids (BCAAs), glutamine, and palmitic acid, abundant nutrient signals of cow´s milk. Furthermore, milk delivers bioactive exosomal microRNAs. After milk consumption, bovine microRNA-29b, a member of the diabetogenic microRNA-29-family, reaches the systemic circulation and the cells of the milk consumer. MicroRNA-29b downregulates branched-chain α-ketoacid dehydrogenase, a potential explanation for increased BCAA serum levels, the metabolic signature of insulin resistance and type 2 diabetes mellitus (T2DM). In non-obese diabetic mice, microRNA-29b downregulates the anti-apoptotic protein Mcl-1, which leads to early β-cell death. In all mammals except Neolithic humans, milk-driven mTORC1 signaling is physiologically restricted to the postnatal period. In contrast, chronic hyperactivated mTORC1 signaling has been associated with the development of age-related diseases of civilization including T2DM. Notably, chronic hyperactivation of mTORC1 enhances endoplasmic reticulum stress that promotes apoptosis. In fact, hyperactivated β-cell mTORC1 signaling induced early β-cell apoptosis in a mouse model. The EPIC-InterAct Study demonstrated an association between milk consumption and T2DM in France, Italy, United Kingdom, Germany, and Sweden. In contrast, fermented milk products and cheese exhibit an inverse correlation. Since the early 1950´s, refrigeration technology allowed widespread consumption of fresh pasteurized milk, which facilitates daily intake of bioactive bovine microRNAs. Persistent uptake of cow´s milk-derived microRNAs apparently transfers an overlooked epigenetic diabetogenic program that should not reach the human food chain.
... It was therefore observed that the human lipoprotein receptor related protein (LRP) showed similarities and differences at several positions in all the 11members. Computation of amino acid composition of each human (LRP) sequences using Protparam tool (see Table 2) indicated very high percentage of leucine, glycine and Serine (with values ranges from 6-12.1%) in almost all the proteins as compare with other amino acids, this invariably may suggested that these amino acids could have a major role as nucleation centers in the folding and evolution of the LRP family [16], while high percentage of these amino acids could account for the stability of the LRP triple helical structure, since incorporation of large amino acids can cause steric hinderance [17]. Also, increase in the proportion of amino acids like asparagine and serine as seen in LRP4, LRP5, LRP6 and LRP10 may aid in processes like cell-cell adhesion, migration and cell signaling properties of these proteins. ...
In this study, 11members of human low density lipoprotein receptor-related protein (LRP)
sequences were retrieved from UniProtKB/ SWISS-PROT protein database and analyzed for information about their structural, functional and phylogenetic features. This was achieved by using many established biocomputational tools which was available at their latest version. This study shows that LRP 12 and 3 are closely related with LRP8 being their nearest neighbor. In all, it was observed that there were very low possession of certain essential amino acid like glycine, proline and a very high aliphatic in all the LRP
family. Considering the evolutionary history, functional domains, high aliphatic index, overall proportion of glycine and proline and the established role of one (LRP8) of this closely related LRP in diseases, it is thus predicted that the other closely related LRP3 and 12 molecules may be important candidate in investigating the aetiopathology of Myocardial infarction1diseases or other heart related disorder.
Recent studies have shown that biodegradable nanoparticles can be efficiently prepared with polymerization of N-carboxyanhydrides-induced self-assembly (NCA-PISA). However, thus far, the effect of chiral monomer ratio on such NCA-PISA formulations and the resulting nanoparticles has not yet been fully explored. Herein, we show, for the first time, that the morphology, secondary structure, and biodegradation rate of PISA nanoparticles can be controlled by altering the chiral ratio of the core-forming monomers. This chirality-controlled PISA (CC-PISA) method allowed the preparation of nanoparticles that are more adjustable and applicable for future biomedical applications. Additionally, the complex secondary peptide structure (ratio of α-helix to β-sheet) and π-π stacking affect the polymer self-assembly process. More specifically, a PEG45 macro-initiator was chain-extended with l- and d-phenylalanine (l- and d-Phe-NCA) in various molar ratios in dry THF at 15 wt%. This ring-opening polymerization (ROP) allowed the preparation of homo- and hetero-chiral Phe-peptide block copolymers that self-assembled in situ into nanoparticles. For homo-chiral formulations, polymers self-assembled into vesicles once a sufficiently high phenylalanine degree of polymerization (DP) was obtained. Hetero-chiral formulations formed larger nanoparticles with various morphologies and, much to our surprise, using an equal enantiomer ratio inhibited PISA and led to a polymer solution instead. Finally, it was shown that the enzymatic biodegradation rate of such PISA particles is greatly affected by the polymer chirality. This PISA approach could be of great value to fabricate nanoparticles that exploit chirality in disease treatment.
Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv ( Mtb- Rv) and MRA_3031 in M. tuberculosis H37Ra ( Mtb- Ra). IlvCs are essential in Mtb- Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb -Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb- Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb- Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb- Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.
The changes of metabolite profiles in preterm birth have been demonstrated using newborn screening data. However, little is known about the holistic metabolic model in preterm neonates. The aim was to investigate the holistic metabolic model in preterm neonates. All metabolite values were obtained from a cohort data of routine newborn screening. A total of 261 758 newborns were recruited and randomly divided into a training subset and a testing subset. Using the training subset, 949 variates were considered to establish a logistic regression model for identifying preterm birth (<37 weeks) from term birth (≥37 weeks). Sventy-two variates (age at collection, TSH, 17α-OHP, proline, tyrosine, C16:1-OH, C18:2, and 65 ratios) entered into the final metabolic model for identifying preterm birth from term birth. Among the variates entering into the final model of PTB [Leucine+Isoleucine+Proline-OH)/Valine (OR=38.36], (C3DC+C4-OH)/C12 (OR=15.58), Valine/C5 (OR=6.32), [Leucine+isoleucine+Proline-OH)/Ornithine (OR=2.509)], and Proline/C18:1 (OR=2.465) have the top five OR values, and [Leucine+Isoleucine+Proline-OH)/C5 (OR=0.05)], [Leucine+Isoleucine+Proline-OH)/Phenylalanine (OR=0.214)], proline/valine (OR=0.230), C16/C18 (OR=0.259), and Alanine/free carnitine (OR=0.279) have the five lowest OR values. The final metabolic model had a capacity of identifying preterm infants with >80% accuracy in both the training and testing subsets. When identifying neonates ≤32 weeks from those >32 weeks, it had a robust performance with nearly 95% accuracy in both subsets. In summary, we have established an excellent metabolic model in preterm neonates. These findings could provide new insights for more efficient nutrient supplements and etiology of preterm birth.
Branched chain amino acids (BCAAs) are building blocks for all life-forms. We review here the fundamentals of BCAA metabolism in mammalian physiology. Decades of studies have elicited a deep understanding of biochemical reactions involved in BCAA catabolism. In addition, BCAAs and various catabolic products act as signaling molecules, activating programs ranging from protein synthesis to insulin secretion. How these processes are integrated at an organismal level is less clear. Inborn errors of metabolism highlight the importance of organismal regulation of BCAA physiology. More recently, subtle alterations of BCAA metabolism have been suggested to contribute to numerous prevalent diseases, including diabetes, cancer, and heart failure. Understanding the mechanisms underlying altered BCAA metabolism and how they contribute to disease pathophysiology will keep researchers busy for the foreseeable future.
Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA complemented with Mtb-Ra ilvA construct. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Enhanced IlvA expression was observed at 48 and 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).
Background:
Carbonic anhydrase I (CA I), a major cecal bacterial antigen, improves inflammatory bowel disease (IBD) symptoms in a murine model. The aim of this study was to identify the responsible epitope region within the CA I protein and evaluate its effect on inflammation using a murine IBD model.
Methods:
Candidate peptides within the CA I protein sequence that interact with major histocompatibility complex class II were chosen and their immune responses were evaluated using mesentery lymph nodes (MLNs) from a CD4CD25 T-cell transfer murine colitis model. Mice were treated with regulatory dendritic cells (Reg-DCs)-pulsed CA I peptide. We assessed their clinical signs, histopathology, induction of cytokines and transcription factors, and generation of CD103CD11c dendritic cells and regulatory T cells (Tregs).
Results:
We identified 4 candidate epitope peptides of CA I. Among these, Reg-DCs pulsed with CA I 58-73 peptide (Reg-DCsCA I 58-73) alone ameliorated colitis. Reg-DCsCA I 58-73-treated mice showed higher mRNA expression levels of forkhead box protein 3, aldehyde dehydrogenase family 1a2, transforming growth factor-β, and interleukin (Il)10, when compared with lower mRNA expression of retinoic acid-related orphan receptor gamma and Il17a in MLNs. Compared with control mice, these mice also showed higher numbers of Foxp3CD4CD25 Tregs and CD103CD11c dendritic cells in MLNs and colon. Administration of Reg-DCsCA I 58-73 induced antigen-specific Tregs in MLNs of colitic mice.
Conclusions:
CA I 58-73 peptide induces antigen-specific therapeutic effect in a murine IBD model using Reg-DCs, indicating that CA I 58-73 is a candidate epitope for IBD immunotherapy.
Even among its detractors [1], the subject of artificial intelligence (AI) is credited with advances in technique in non-numerical computing. As the value of this type of computing in physics in particular and physical sciences in general is now beyond doubt, it should be useful to see how work in physical sciences and in AI can be related. To begin, it is reasonable to ask two questions:
(ql)
What is AI?
(q2)
What implications does AI have for physical problems (and vice versa)?
Studies on the chiroptical properties of polypeptides containing aromatic side-chain chromophores are in general complicated by strong overlapping contributions from peptide and side-chain chromophores. Nevertheless such investigations are essential in order to know the details of the aromatic contributions to the optical activity as related to the structure, especially in connection with aromatic Cotton effects observed in CD spectra of proteins. The CD bands associated with tyrosyl-, tryptophanyl-, histidyl-, and phenyl- alanyl-residues in proteins are very sensitive probes of local conformations and could yield valuable structural information.
Apolipoprotein A-1 [apo A-1], the major protein of high density lipoproteins [HDL] [1], is an activator of the plasma enzyme lecithin, cholesterol acyltrans-ferase [LCAT]. This enzyme transfers fatty acids from lecithin to cholesterol in high density lipoproteins and is responsible for the formation of nearly all plasma cholesteryl esters [2]. It has been suggested that the activation of LCAT by apo A-1 is due in part to its amphiphilic character. Synthetic models of apolipopeptides with a primary structure different from that of apo A–I, but having a similar amphiphilic character and helical potential, also activate LCAT [3,4]. The role of apo A-1 in the mechanism of LCAT activation is important but poorly understood.
The introduction of a simple and efficient peptide synthesis methodology in the early 1960’s (1) put a powerful research tool at the disposal of biologists and protein chemists alike. Thus, it was immediately feasible to synthesize not only biologically active peptides, but also discrete segments of proteins. It was obvious from the start, also, that this technology could be coupled with longstanding immunologic procedures whereby small molecules can be used as antigenic materials (2). Indeed, the approach was soon applied to a number of proteins in a quest to find what features made them antigenic (3). It is fair to say, though, that the full capacity of the combined immunologic and synthetic techniques was never exploited during the 1960’s and 1970’s. Since then advances in the field of DNA sequencing have provided a plethora of sequence information about proteins, real and putative, many of which have never been isolated, never mind characterized. The sequence data can be utilized in a variety of ways, especially in conjunction with computer-aided predictive schemes that allow a degree of meaningful conjecture about the three-dimensional structure of a given sequence. Among the ends to which DNA/protein sequence data can be put is the chemical synthesis of peptides corresponding to portions of the protein.
Amino acid biosynthesis is one of the cardinal events of carcinogenesis that has not been investigated in urothelial carcinoma (UC). By data mining a published transcriptomic database of UCs of urinary bladder (UBUCs) (GSE31684), we identified branched-chain amino acid transaminase 1 (BCAT1) as the most significantly stepwise upregulated gene during tumor progression among those associated with amino acid biosynthetic process (GO:0008652). Accordingly, we analyzed BCAT1 transcript and protein expression with their clinicopathological significance.
We used real time RT-PCR to detect BCAT1 transcript levels in 20 UCs of upper tract (UTUCs) and 20 UBUCs, respectively. Immunohistochemical study was performed to determine BCAT1 protein expression in 340 UTUCs and 295 UBUCs. Higher BCAT1 transcript levels were associated with higher pT status in both groups (p<0.05). BCAT1 protein overexpression was also significantly associated with adverse clinicopathological features, e.g., advanced pT stage, nodal metastasis, high pathological grade and etc. (p<0.05). BCAT1 overexpression predicted worse disease-specific survival and metastasis-free survival in both univariate and multivariate analyses (p≤0.001).
BCAT1 overexpression is associated with advanced tumor status, and implies adverse clinical outcomes of UCs, suggesting its role in tumor progression and could serve as a prognostic biomarker and a novel therapeutic target in UC. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The tenet of structural biology that function follows form had its seeds in the monograph by C. B. Anfinsen, The Molecular Basis of Evolution (Anfinsen, 1959), wherein he stated “Protein chemists naturally feel that the most likely approach to the understanding of cellular behavior lies in the study of structure and function of protein molecules.” The achievement of protein crystallography over the past 30 years has confirmed this view whereby the description of the structure and function of proteins is now frequently understood at the atomic level.
The conformational properties of methyl esters were examined in anhydrous and aqueous organic solvents. This study demonstrates that these oligomers may exist in predominantly β-associated or unordered conformations depending upon the solvent. The β-structure, which appears at the pentamer in ethylene glycol (EG) and trifluoroethanol (TFE)-water (20:80 v/v), could be disrupted by dilution or by increasing the temperature. The stability of the β-structures was found to be lower than those formed by oligopeptides derived from l-isoleucine and l-valine and comparable to those formed by oligo-l-methionines.
Based on the helix and-sheet and the-turn conformational parameters, and and , of the 20 amino acids, we propose a new 3D graphical representation of protein sequence without circuit or degeneracy, which may reflect the innate structure of the protein sequence. Then the numerical characterizations of protein graphs, the leading eigenvalues of the L/L matrices associated with the graphical curves for protein sequences, was utilized as descriptors to analyze the similarity/dissimilarity of the nine ND5 protein sequences.
At the forefront of medicine, gene therapy provides an effective way to treat a range of diseases by regulating defective genes at the root of the disease. Short interfering RNAs (siRNAs) hold great promise as therapeutic agents in this domain; however, intracellular delivery remains a major obstacle to clinical application of therapeutic siRNAs. Here we report a peptide designed to mediate siRNA delivery. This peptide, C6M1, is rationally designed to promote the endosomal escape ability of an existing peptide sequence. Formed C6M1/siRNA nanoscale complexes are able to deliver siRNA into cells and induce specific gene knockdown with low toxicity. The increased membrane disruption ability at acidic condition of the peptide with tryptophan residue substitution may contribute to the enhanced gene silence efficacy. Intratumoral injection of the complexes results in a marked reduction of tumor growth through downregulation of antiapoptotic Bcl-2 protein in mice. In addition, the C6M1/siRNA complex was proven safe at transfection concentration by cytotoxicity assay. These results demonstrate that the C6M1/siRNA complex is a potent system for efficient gene delivery in vitro and in vivo.
Methods for describing a discrete number of conformational states of amino acid residues in proteins are presented and used to investigate the topography of chain folding. The relative importance of short-range, medium-range and long-range interactions is discussed in the light of an analysis of the conformational states for the different amino acid residues in eight proteins of known structure. A prediction algorithm, which assigns four states to each residue of a protein chain (α-helix, extended structure, bend, or coil), has been developed from a consideration of both short- and medium-range interactions and applied to thirteen proteins of known three-dimensional structure. The prediction algorithm is simple to apply, and the assignment of α-helix and extended structure is considerably better than in most other predictive schemes. The prediction of chain reversal or bend regions was also better than with previous algorithms, but these assignments were not as good as those for α-helix and extended structure. The motivation for the development of this algorithm is not only to demonstrate the relative importance of short- and longer-range interactions but, more important, to begin to develop procedures for obtaining an approximate starting conformation for subsequent energy minimization to predict the three-dimensional structure of a protein. This procedure, as well as various other methods for the prediction of the backbone topography and conformational states of residues in proteins from the amino acid sequence, have been reviewed and evaluated by comparing the success of the methods to the success expected from a random assignment of conformational states.