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Ligand interaction scan: A general method for engineering ligand-sensitive protein alleles
Abstract
The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).
... [14,46] The region that is most frequently used for TC environment design is a protein loop: the motif is incorporated either into a protein-chain turn, or into a domain-connecting region, by insertion of a TC sequence or by replacement of the existing amino acids (Figure 2 C). [14,[47][48][49][50][51][52][53][54][55][56][57][58][59][60][61] This approach is often used when the protein structure is known, but it is possible to identify exposed loops by bioinformatic prediction and experi- Figure 1. Zn II inhibition of TC motif labeling with biarsenicals (BiAs-EDT 2 ). ...
... It should be noted that the fluorescence of biarsenical TC loops varies significantly, even within a single protein, and it strongly depends on the surrounding amino acids. [47] Another type of biarsenical coordination environment is built on protein splits and bipartite structures. There are several possibilities for linking a probe by using such a coordination mode (Figure 2 D). ...
... Testing of different mutants that contained the TC motif showed that insertions at only slightly different positions can have significantly different effects on activity. It should be noted that the TC motif alone causes re-duced catalytic capabilities (V max , K m ) when compared to WT. [47] A different approach to find regulatory sites was used in the case of T-cell protein tyrosine phosphatase (PTP). Insertions were placed in loops that had no conserved amino acids or secondary structure elements. ...
The fluorescent modification of proteins (with genetically encoded low-molecular-mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent-labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6- to 12-residue), appropriately spaced, tetracysteine sequence (-CCXXCC-); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact-cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein-protein interaction studies, protein stability, and aggregation studies.
... It enables the sitedirected insertion of any length of foreign DNA into an existing sequence, and its implementation in the procedure described here also enables a rapid verification of the presence of the inserted sequence. This procedure has proved useful for inserting a ligandbinding domain into a target protein (12). Yet, it may readily be applied to insertions of functional domains, cleavage sites, tags, or other desired sequences into a target gene, or adding restriction sites and regulatory elements into a target plasmid. ...
... Using the modified inverse PCR procedure described herein, we recently developed a novel, general, and simple procedure for engineering small-molecule ligand-regulated forms of any protein (12). The Ligand Interaction Scan (LIScan) method involves insertion of a chemical-genetic "switch," comprising a genetically encoded peptide module (a tetracysteine-containing hexapeptide) that binds with high affinity to a small-molecule ligand (the cell-permeable biarsenical fluorescein derivative FlAsH, (13)) into a given protein. ...
... The insertion position(s) are selected empirically to confer ligand-dependent modulation of activity. Ligand-regulated mutants may then be expressed in cells or inserted genomically, wherein they can be regulated by ligand administration (12). ...
Functional analysis of a protein of interest, by generation of functional alterations in a target protein, often requires the performance of site-directed mutagenesis within the gene sequence. These manipulations are usually performed using "cut and paste" techniques, combined with PCR. Here we describe a simple and general procedure to specifically insert a DNA fragment into any site within a given DNA sequence. We demonstrate this insertional mutagenesis by describing the insertion of a tetracysteine (4C) hexapeptide-encoding sequence into the coding sequence of the antibiotic hydrolyzing enzyme TEM-1 beta-lactamase. This procedure could also be applied to insert different DNA sequences or to replace, or delete, existing fragments in a given gene. We have recently used this procedure to develop a general method (ligand interaction scan - LIScan) to generate ligand-regulated proteins.
... This method, which we term the ligand interaction scan (LIScan), calls for the generation of mutants whose activity is altered upon addition of a small-molecule ligand. The mutants are prepared by multiple insertions of short peptides which are composed of two pairs of vicinal Cys residues separated by a Pro and Gly linker (4C sequence), to the examined proteins [15]. This peptide is recognized specifically and with high affinity by a cell-permeable molecule, termed FlAsH [16], which modulates the activity of some of the mutated proteins and allows their study. ...
... This peptide is recognized specifically and with high affinity by a cell-permeable molecule, termed FlAsH [16], which modulates the activity of some of the mutated proteins and allows their study. We previously used the 4C peptide and the FlAsH to show that they could be used to switch the activity of a target protein on or off [15]. Due to the unknown nature of the biological function of ERK8, we postulated that its function and localization can be determined by LIScan. ...
... First strand cDNA synthesis was done using MMLV-RT and random DNA hexamers as primers (Promega, Madison, WI). ERK8 mutants containing the 4C peptide insertion were generated and characterized as described before [15]. Briefly, a modified inverse PCR procedure [17] was applied to insert the 4C at desired locations within a plasmid containing the ERK8 coding sequence. ...
ERK8 is the most recent addition for the MAPK family, and its mechanism of activation and function are not yet known, mainly due to the lack of any known physiological stimulator. In this report, we describe the preparation of reagents for the use of a novel method, the ligand interaction scan (LIScan), to study the function of this protein kinase. We generated a set of mutants of ERK8, and identified inhibited as well as stimulated forms. By specifically inhibiting or stimulating the mutants of ERK8, we show that the ERK8-induced inhibition of proliferation is altered. Moreover, we used the developed mutants to show for the first time that ERK8 translocates to the nucleus upon activation. The use of methods such as the ligand interaction scan may thus promote the analyses of the functions of uncharacterized proteins such as ERK8, and possibly help in controlling the activity of target proteins in various experimental systems and applications.
... Could there exist a means to introduce de novo "activation loops" in enzymes for which other chemical-rescue strategies have not been developed? A recent report has shown that insertion of a short peptide into a protein domain can indeed be used to generate an activatable enzyme variant [33]. This study utilized the cell-permeable small-molecule fluorescein arsenical hairpin binder (FlAsH), which is known to bind specifically to peptides containing tetra-cysteine motifs [34,35]. ...
... FlAsH has no significant affinity for wild-type TEM-1 (which contains no CCPGCC motifs), but binds to and modulates the activity of mutants containing CCPGCC. The authors identified several FlAsH-responsive mutants, most of which are inhibited by FlAsH binding [33]. By contrast, the TEM-1 mutant with a CCPGCC insertion at amino acid 215 (4C-215 TEM-1) is specifically activated by FlAsH. ...
Cell-permeable small molecules that are capable of activating particular enzymes would be invaluable tools for studying protein function in complex cell-signaling cascades. But, is it feasible to identify compounds that allow chemical-biology researchers to activate specific enzymes in a cellular context? In this review, we describe some recent advances in achieving targeted enzyme activation with small molecules. In addition to surveying progress in the identification and targeting of enzymes that contain natural allosteric-activation sites, we focus on recently developed protein-engineering strategies that allow researchers to render an enzyme of interest "activatable" by a pre-chosen compound. Three distinct strategies for targeting an engineered enzyme are discussed: direct chemical "rescue" of an intentionally inactivated enzyme, activation of an enzyme by targeting a de novo small-molecule-binding site, and the generation of activatable enzymes via fusion of target enzymes to previously characterized small-molecule-binding domains.
... The idea to allosterically inhibit an enzyme using a precisely localized biarsenical probe was simultaneously developed for TEM b-lactamase and T-cell protein tyrosine phosphatase (TCPTP). 238,239 In both cases it was found that multiple locations must be scanned for the TC tag in order to efficiently deactivate the enzyme with a biarsenical probe. Also, compared to the WT, the TC tagged enzyme exhibited slightly reduced catalytic activity. ...
Fluorescent modification of proteins of interest (POI) in living cells is desired to study their behaviour and functions in their natural environment. In a perfect setting it should be easy to perform, inexpensive, efficient and site-selective. Although multiple chemical and biological methods have been developed, only a few of them are applicable for cellular studies thanks to their appropriate physical, chemical and biological characteristics. One such successful system is a tetracysteine tag/motif and its selective biarsenical binders (e.g. FlAsH and ReAsH). Since its discovery in 1998 by Tsien and co-workers, this method has been enhanced and revolutionized in terms of its efficiency, formed complex stability and breadth of application. Here, we overview the whole field of knowledge, while placing most emphasis on recent reports. We showcase the improvements of classical biarsenical probes with various optical properties as well as multifunctional molecules that add new characteristics to proteins. We also present the evolution of affinity tags and motifs of biarsenical probes demonstrating much more possibilities in cellular applications. We summarize protocols and reported observations so both beginners and advanced users of biarsenical probes can troubleshoot their experiments. We address the concerns regarding the safety of biarsenical probe application. We showcase examples in virology, studies on receptors or amyloid aggregation, where application of biarsenical probes allowed observations that previously were not possible. We provide a summary of current applications ranging from bioanalytical sciences to allosteric control of selected proteins. Finally, we present an outlook to encourage more researchers to use these magnificent probes.
... Our method shares many features with these two strategies, but the ability to scan Z through a protein sequence should make it much more broadly applicable than the bump-and-hole strategy and the chemoselectivity of "click" reactions should allow it to be applied in vivo, unlike the tethering strategy. We note examples of similar strategies using tetracysteine motifs and bisarsenical compounds, but these methods have not been broadly applied 519,520 . ...
Proteins play important roles in biological processes including muscle movement, initiating immune responses, and memory formation. Full understanding of protein function requires determining its structure, assignment of function, and elucidation of interactions with other proteins or metabolites. Protein modification is useful for installing chemical entities that allow researchers to specifically probe function, attach fluorophores for localization, covalently trap protein interactions, or perform affinity enrichment from a complex milieu. Installation of functional groups otherwise not accessible from canonical protein expression requires a toolbox of protein modification strategies. We have developed strategies for using a combination of techniques to functionalize proteins, including E. coli aminoacyl transferase (AaT) mediated N-terminal protein modification, Amber codon suppression for incorporation of unnatural amino acids (Uaas), auxotrophic strain incorporation of Uaas, and click chemistry reactions. A chemoenzymatic approach is used with AaT mediated N-terminal transfer of an azide functionality and subsequent click- chemistry to fluorescently label protein N-termini. We also use AaT to install a unique N-terminal protein semi-synthesis ligation site. Additionally, Amber codon suppression is used to site-specifically incorporate Uaa side chains with click-chemistry handles, intrinsic fluorophores, and photocrosslinkers into proteins. Click-chemistry is used with Amber codon suppression to control protein function in a highly selective manner. We also use auxotrophic strain incorporation to globally replace a natural amino acid with an isostere Uaa to introduce azide and alkyne functional groups. Protein multifunctionalization is achieved by combining these modification methods in a single expressed protein. Effectively achieving this combination has required careful consideration of both molecular biology and chemical reactivity. We demonstrate the combination of these techniques to label alpha-synuclein, a protein which is involved in neurodegenerative diseases. Moreover, the protocols for multiple labeling that we have developed should be generally practicable for a variety of proteins.
... Nevertheless, this contributes not just to drug discovery research, but also to protein engineering. Actually, some β-lactamases, such as PenP and TEM-1, which were used in this study, become popular protein models for protein engineering researches [56][57][58][59][60][61][62]. In addition, these information are particularly important and helpful for the work in this project. ...
... This is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. Scientists demonstrated the method with TEM-1 -lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH) [149]. Molecular strategies are being developed for the use of cyclic peptides in forward and reverse genetics experiments [150]. ...
Chemical biology (CB) is a scientific discipline spanning the fields of chemistry and biology involving the application of chemical techniques and tools, often natural products or small chemical compounds produced through synthetic chemistry, to the study and manipulation of biological systems. CB is providing new tools for deciphering protein modification and activity. Designer small molecules as probes of protein and cellular function are becoming the method of choice for careful profiling and development of biological processes in areas such as drug discovery, neurochemistry and molecular genetics. CB studies consists of three methodologies, chemical libraries with small molecules, high-throughput screening, and computational database. Combinatorial synthetic methods combined with high-throughput screening provided large numbers of potential lead compounds. CB reach is expanding into a vide range of fields and there is need to introduce CB to professional chemists working in biotechnology, pharmaceutical, agrochemical and other relevant sectors. Inorganic/organometallic/bioinorganic chemistry-chemical biology interface, biomolecular interactions (protein-protein, protein-carbohydrate), fluorescent quantum dots, biocongugation techniques, boronic acid-based chemosensors, click chemistry and artificial enzymes are described. CB aspects in aging, microfluidics, neurodegenerative diseases, human immunodeficiency virus as well as PROteolysis TArgeting Chimeric moleculeS (PROTACS) are reported. Research on the structural and energetic factors that distinguish specific protein interfaces should be strengthened. Exploration of protein networks should be actively pursued. Chemical biology new technologies may accelerate the discovery of still elusive cures to neurodegenerative diseases.
... This is the first study to insert two non-contiguous 4C tags into a protein sequence. Examples of single insertions include the host-encoded cellular prion protein (PrP C ) (44); the GAG protein in HIV-1 (45); TEM-1 beta-lactamase (46) and the α(2A)-adrenergic G protein-coupled receptor (47). ...
During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)-containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse-chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time-lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43-GFP-4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno-labeled endogenous Cx43-expressing cells. Photo-oxidation of ReAsH-labeled Cx43-GFP-4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse-chase labeling and single label time-lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit.
Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a “clickable magic bullet” strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.
Ein neuer chemischer In-vivo-Verknüpfer: xCrAsH, ein dimeres Derivat der Bisarsenitverbindung FlAsH, ermöglicht die hochspezifische, kovalente Verknüpfung zweier Proteine, die je eine zwölf Aminosäuren lange Markierung tragen. Dieses induzierbare und (durch Zugabe von Dithiolen) reversible System kann für die Detektion und Manipulation von Protein-Protein-Wechselwirkungen in vitro und in lebenden Zellen verwendet werden (siehe Bild).
Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.
Organometallic compounds are renowned for their remarkable applications in the field of catalysis, but much less is known about their potential in chemical biology. Indeed, such compounds have long been considered to be either unstable under physiological conditions or cytotoxic. As a consequence, little attention has been paid to their possible utilisation for biological purposes. Because of their outstanding physicochemical properties, which include chemical stability, structural diversity and unique photo- and electrochemical properties, however, organometallic compounds have the ability to play a leading role in the field of chemical biology. Indeed, remarkable examples of the use of such compounds-notably as enzyme inhibitors and as luminescent agents-have recently been reported. Here we summarise recent advances in the use of organometallic compounds for chemical biology purposes, an area that we define as "organometallic chemical biology". We also demonstrate that these recent discoveries are only a beginning and that many other organometallic complexes are likely to be found useful in this field of research in the near future.
We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15-HEAT repeat protein PR65A. PR65A is one of a special class of modular non-globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one-dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the α-helical HEAT array of PR65A without disrupting the structure or stability. When the linear tetracysteine motif CCPGCC was used, FlAsH fluorescence reported globally on the folding of the protein. When the tetracysteine motif was displayed in bipartite mode through the engineering of pairs of cysteines on adjacent HEAT repeats, FlAsH fluorescence became a reporter of local conformation and of oligomerisation. Thus, by designing FlAsH-binding sites at different locations along the repeat array one can interrogate specific properties of PR65A, paving the way for structure-function analysis of this protein both in vitro and in the cell.
A FlAsH of potential: the specific binding of the biarsenical probe to the tetracysteine motif has matured as a tool for cell biology studies. Combining two such binders in one probe generates a useful reporter of protein dimerization events. The current state of art and the perspective for future developments are highlighted.
Protein tyrosine phosphatase 1B (PTP1B) is a key regulator of the insulin-receptor and leptin-receptor signaling pathways, and it has therefore emerged as a critical antitype-II-diabetes and antiobesity drug target. Toward the goal of generating a covalent modulator of PTP1B activity that can be used for investigating its roles in cell signaling and disease progression, we report that the biarsenical probe FlAsH-EDT(2) can be used to inhibit PTP1B variants that contain cysteine point mutations in a key catalytic loop of the enzyme. The site-specific cysteine mutations have little effect on the catalytic activity of the enzyme in the absence of FlAsH-EDT(2). Upon addition of FlAsH-EDT(2), however, the activity of the engineered PTP1B is strongly inhibited, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. We show that the cysteine-rich PTP1B variants can be targeted with the biarsenical probe in either whole-cell lysates or intact cells. Together, our data provide an example of a biarsenical probe controlling the activity of a protein that does not contain the canonical tetra-cysteine biarsenical-labeling sequence CCXXCC. The targeting of "incomplete" cysteine-rich motifs could provide a general means for controlling protein activity by targeting biarsenical compounds to catalytically important loops in conserved protein domains.
We report that the activity of a rationally engineered protein tyrosine phosphatase (PTP) mutant can be fully rescued by the addition of the biarsenical fluorescein derivative FlAsH, a compound that does not affect the activity of wild-type PTPs.
A general method was developed for the discovery of protease-activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease-2 substrate linker to a vascular endothelial growth factor-binding peptide and sorted using a two-stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease-mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100-fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.
A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.
Small molecules that can be used to turn off the activities of specific cellular proteins are essential tools for chemical biology. Few such compounds are known, however, and they are particularly difficult to identify for members of large protein families. Here, we present a method for insertion of a chemical "off switch" into a catalytically essential loop region (the "WPD loop") of a protein tyrosine phosphatase (PTP). Using a combination of point mutations and amino acid insertions, we have engineered variants of T-cell PTP (TCPTP) that possess cysteine-rich WPD loops. The engineered WPD loops, which contain sequences that appear in no wild-type PTP, confer upon TCPTP the ability to bind a cell-permeable small molecule (the biarsenical fluorescein derivative, FlAsH) that is not an inhibitor of wild-type PTPs. We have identified sites in TCPTP's WPD loop that can be modified to display FlAsH-binding cysteine residues without disrupting TCPTP's inherent PTP activity, as assayed with either small-molecule or phosphorylated-peptide PTP substrates. Upon addition of the FlAsH ligand, however, the activities of the mutants drop dramatically. Inhibition of the FlAsH-sensitized TCPTP mutants is rapid and specific; and strong FlAsH sensitivity was observed in mutants that contain as few as two cysteine point mutations in their engineered WPD loops. Our results show that relatively conservative substitutions can be used to engineer precise small-molecule control of PTP activity. Moreover, since all known classical PTPs utilize the WPD-loop mechanism targeted in this study, it is likely that a substantial fraction of the PTP superfamily can be sensitized through an analogous approach.
The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.
High level expression of TEM beta-lactamase results in the accumulation of precursor and mature protein in the insoluble fraction of Escherichia coli. The mature polypeptide is sequestered in protein aggregates (inclusion bodies) located within the periplasmic space whereas the insoluble precursor is present in the cytoplasm. With the native beta-lactamase, aggregation is observed when the rate of expression exceeds 2.5% of the total protein synthesis rate. Substitution of the native signal sequence with the outer membrane protein A (OmpA) leader peptide results in extensive aggregation of only the mature protein. Furthermore, for OmpA-beta-lactamase, the accumulation of mature insoluble protein is independent of the rate of protein synthesis. These observations cannot be accounted by the kinetics of export of the OmpA-beta-lactamase and the native precursor, therefore suggesting that the signal sequence affects the conformation of the newly secreted mature polypeptide and in turn, the folding pathway. Previously, we have shown that the aggregation of the mature protein secreted using its own signal sequence can be inhibited by growing the cells in the presence of non-metabolizable sugars such as sucrose (Bowden, G., and Georgiou, G. (1988) Biotechnol. Prog. 4, 97-101). We show here that this phenomenon is not related to osmotic effects, changes in beta-lactamase translation or precursor processing. It follows that the addition of sugars exerts a direct effect on the in vivo pathway of aggregation and folding, in analogy with the well characterized effect of sugars in vitro.
We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification
by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that
include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template
DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to
be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the
desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of
most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found
that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the
newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene
sequence.
beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 A crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1) and (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, K(I) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this potent class of beta-lactamase inhibitors.
Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.
Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.
Recombinant proteins containing four cysteines at the i,i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein.
This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the
tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility
in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly
modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently
stained by small nonfluorescent dye molecules added from outside the cells.
We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
University Microfilms order no. 3104153. Thesis (Ph. D.)--Stanford University, 2003. Includes bibliographical references.
Dimerization and oligomerization are general biological control mechanisms contributing to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. Cell permeable, synthetic ligands were devised that can be used to control the intracellular oligomerization of specific proteins. To demonstrate their utility, these ligands were used to induce intracellular oligomerization of cell surface receptors that lacked their transmembrane and extracellular regions but contained intracellular signaling domains. Addition of these ligands to cells in culture resulted in signal transmission and specific target gene activation. Monomeric forms of the ligands blocked the pathway. This method of ligand-regulated activation and termination of signaling pathways has the potential to be applied wherever precise control of a signal transduction pathway is desired.
Anew insertion method for probing protein functional organization was developed. The method relies on the random insertion
of transposon Tn4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene. This results in pentapeptide
insertions randomly distributed in the target protein. Characterization of 23 pentapeptide insertions in TEM-1 β-lactamase
demonstrated the utility of the method. The phenotypes associated with the mutated β-lactamase proteins equated both with
the sorts of local peptide structures in which the pentapeptide insertions occurred and their position in the three-dimensional
structure of the enzyme.
Recombinant proteins containing four cysteines at the i, i + 1, i + 4, and i + 5 positions of an alpha helix were fluorescently labeled in living cells by extracellular administration of 4',5'-bis(1,3, 2-dithioarsolan-2-yl)fluorescein. This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.
Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.
New chemical methods that use small molecules to perturb cellular function in ways analogous to genetics have recently been developed. These approaches include both synthetic methods for discovering small molecules capable of acting like genetic mutations, and techniques that combine the advantages of genetics and chemistry to optimize the potency and specificity of small-molecule inhibitors. Both approaches have been used to study protein function in vivo and have provided insights into complex signaling cascades.
Protein splicing is a naturally occurring process in which an intervening intein domain excises itself out of a precursor polypeptide in an autocatalytic fashion with concomitant linkage of the two flanking extein sequences by a native peptide bond. We have recently reported an engineered split VMA intein whose splicing activity in trans between two polypeptides can be triggered by the small molecule rapamycin. In this report, we show that this conditional protein splicing (CPS) system can be used in mammalian cells. Two model constructs harboring maltose-binding protein (MBP) and a His-tag as exteins were expressed from a constitutive promoter after transient transfection. The splicing product MBP-His was detected by Western blotting and immunoprecipitation in cells treated with rapamycin or a nontoxic analogue thereof. No background splicing in the absence of the small-molecule inducer was observed over a 24-h time course. Product formation could be detected within 10 min of addition of rapamycin, indicating the advantage of the posttranslational nature of CPS for quick responses. The level of protein splicing was dose dependent and could be competitively attenuated with the small molecule ascomycin. In related studies, the geometric flexibility of the CPS components was investigated with a series of purified proteins. The FKBP and FRB domains, which are dimerized by rapamycin and thereby induce the reconstitution of the split intein, were fused to the extein sequences of the split intein halves. CPS was still triggered by rapamycin when FKBP and FRB occupied one or both of the extein positions. This finding suggests yet further applications of CPS in the area of proteomics. In summary, CPS holds great promise to become a powerful new tool to control protein structure and function in vitro and in living cells.
Chemistry-driven strategies for modifying, controlling and monitoring protein function in vitro and in vivo have attracted widespread interest among chemists in recent years. Several strategies have now emerged that complement standard genetics-based approaches, and they are being increasingly adopted by biologists to address issues in relevant contexts from cells to animals. With the development of these chemical biology tools, we might be approaching a time when detailed quantitative analysis of protein function, to a degree previously available only in reconstituted systems, is attainable in an in vivo setting.
Imaging of biochemical processes in living cells and organisms is essential for understanding how genes and gene products work together in space and time and in health and disease. Such imaging depends crucially on indicator molecules designed to maximize sensitivity and specificity. These molecules can be entirely synthetic, entirely genetically encoded macromolecules, or hybrid combinations, each approach having its own pros and cons. Recent examples from the author's laboratory include peptides whose uptake into cells is triggered by proteases typical of tumors, monomeric red fluorescent proteins and biarsenical-tetracysteine systems for determining the age and electron-microscopic location of proteins.
We have developed a general method of making conditional alleles that allows the rapid and reversible regulation of specific proteins. A mouse line was produced in which proteins encoded by the endogenous glycogen synthase kinase-3 beta (GSK-3beta) gene are fused to an 89 amino acid tag, FRB*. FRB* causes the destabilization of GSK-3beta, producing a severe loss-of-function allele. In the presence of C20-MaRap, a highly specific, nontoxic, cell-permeable small molecule, GSK-3betaFRB* binds to the ubiquitously expressed FKBP12 protein. This interaction stabilizes GSK-3betaFRB* and restores both protein levels and activity. C20-MaRap-mediated stabilization is rapidly reversed by the addition of an FKBP12 binding competitor molecule. This technology may be applied to a wide range of FRB*-tagged mouse genes while retaining their native transcriptional control. Inducible stabilization could be valuable for many developmental and physiological studies and for drug target validation.
- S R Adams
Adams, S.R. et al. J. Am. Chem. Soc. 124, 6063–6076 (2002).
- B Hallet
- D J Sherratt
- F Hayes
Hallet, B., Sherratt, D.J. & Hayes, F. Nucleic Acids Res. 25, 1866–1867 (1997).