Ranjith Muhandiram

University of Toronto | U of T · Department of Molecular Genetics

Misofolding of mammalian prion proteins (PrP) is believed to be the cause of a group of rare and fatal neurodegenerative diseases. Despite intense scrutiny however, the mechanism of the misfolding reaction remains unclear. We perform nuclear Magnetic Resonance and thermodynamic stability measurements on the C-terminal domains (residues 90-231) of t...

Intrinsically disordered proteins play important roles in cell signalling, transcription, translation and cell cycle regulation. Although they lack stable tertiary structure, many intrinsically disordered proteins undergo disorder-to-order transitions upon binding to partners. Similarly, several folded proteins use regulated order-to-disorder trans...

Significance Intrinsically disordered proteins lack persistent folded structure but are abundant and critical for regulatory protein interactions. They represent a challenge to the structure/function paradigm, particularly when significant disorder remains on interaction with partners. Although many disordered protein interactions have been describ...

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible ‘excited’ conformers. Analysis of CPMG data-sets also provides the m...

FF domains are poorly understood protein interaction modules that are present within eukaryotic transcription factors, such as CA150 (TCERG-1). The CA150 FF domains have been shown to mediate interactions with the phosphorylated C-terminal domain of RNA polymerase II (phosphoCTD) and a multitude of transcription factors and RNA processing proteins,...

Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical shift differences between NMR probes in the exchanging stat...

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins us...

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Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded st...

The N terminus of the c-Myc oncoprotein interacts with Bin1, a ubiquitously expressed nucleocytoplasmic protein with features of a tumor suppressor. The c-Myc/Bin1 interaction is dependent on the highly conserved Myc Box 1 (MB1) sequence of c-Myc. The c-Myc/Bin1 interaction has potential regulatory significance as c-Myc-mediated transformation and...

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to invest...

A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on (15)N, natural abundance (13)C samples. The experiment exploits the fact that transfers of magnetization from (15)N to (13)CO and from (15)N to (13)C(alpha) (and back) proceed independently for samples that are not enriched in (13)C. A factor...

X-linked lymphoproliferative disease is caused by mutations in the protein SAP, which consists almost entirely of a single SH2 domain. SAP interacts with the Tyr281 site of the T<-->B cell signaling protein SLAM via its SH2 domain. Interestingly, binding is not dependent on phosphorylation but does involve interactions with residues N-terminal to t...

A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported. The assignment strategy follows from our previously described approac...

New pulse sequences are presented for the measurement of the relaxation of deuterium double quantum, quadrupolar order, and transverse antiphase magnetization in (13)CH(2)D methyl groups of (15)N-, (13)C-labeled, fractionally deuterated proteins. Together with previously developed experiments for measuring deuterium longitudinal and transverse deca...

Multisite magnetization transfer (MMT) studies of metal migration around the C7H7 rings in (η3-C7H7)Re(CO)4 (1), (η3-C7H7)Os(CO)3SnPh3 (2), (η3-C7H7)Re(CO)3PMe3 (3), (η5-C7H7)Re(CO)3 (4), (η5-C7H7)Fe(CO)2SnPh3 (5), (η5-C7H7)Os(CO)2SnPh3 (6), and (η5-C7H7)Ru(CO)2SnPh3 (7) are reported. The possible presence of 1,2-, 1,3-, and/or 1,4-metal shifts in...

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radiu...

The SH2 domain protein SAP/SH2D1A, encoded by the X-linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation-independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified...

The use of a short, three-residue Cu(2+)-binding sequence, the ATCUN motif, is presented as an approach for extracting long-range distance restraints from relaxation enhancement NMR spectroscopy. The ATCUN motif is prepended to the N-termini of proteins and binds Cu(2+) with a very high affinity. Relaxation rates of amide protons in ATCUN-tagged pr...

†Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecul...

Solution NMR studies on the physiologically relevant ligand-free and maltotriose-bound states of maltodextrin-binding protein (MBP) are presented. Together with existing data on MBP in complex with beta-cyclodextrin (non-physiological, inactive ligand), these new results provide valuable information on changes in local structure, dynamics and globa...

The Leu99-->Ala mutant of T4 lysozyme contains a large internal cavity in the core of its C-terminal domain that is capable of reversibly binding small hydrophobic compounds. Although the cavity is completely buried, molecules such as benzene or xenon can exchange rapidly in and out. The dynamics of the unliganded protein have been compared to the...

DNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound full-length p53 transactiva...

Experiments for measuring chemical shift anisotropy (CSA) cross-correlated spin relaxation between pairs of carbonyl spins in 15N,13C-labeled proteins based on differences in relaxation rates of double- and zero-quantum carbonyl coherences are presented. In the case where the carbonyl spins reside on successive residues, information about the inter...

Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even polyproline stretches. The lack of amide protons in such regions complicates assignment, since 1HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the prot...

The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains di...

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We re...

General transcription factor TFIID consists of TATA box–binding protein (TBP) and TBP-associated factors (TAFIIs), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAFII (dTAFII230) binds directly to TBP and inhibits TBP binding to the TATA box. We report h...

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H, 15N, 13C labelled intact and (des2-7) trp repressor (delta 2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulti...

Protein recognition is a key determinant in regulating biological processes. Structures of complexes of interacting proteins provide significant insights into the mechanism of specific recognition. However, studies performed by modifying residues within a protein interface demonstrate that binding is not fully explained by these static pictures. Th...

A triple-resonance pulse scheme is described which records(15)N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a(15)N, (13)C and fractionally deuterated proteinsample and selects for CH(2)D methyl types. The experiment isthus useful in the early stages of the sequential assignment pro...

Pulse sequences are presented for the measurement of 3JC'C gamma and 3JNC gamma scalar couplings for all C gamma containing residues in 15N,13C uniformly labeled proteins. The methods described are based on quantitative J correlation spectroscopy pioneered by Bax and co-workers [Bax et. al. (1994) Methods Enzymol., 239, 79-105]. The combination of...

Pulse sequences are presented for the measurement of3JCC and3JNC scalar couplings for allC containing residues in15N,13C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of 3JCC and3JNC scalar couplin...

Pulse sequences are presented for the measurement of (3)J(C'C gamma) and (3)J(NC gamma) scalar couplings for all C-gamma containing residues in N-15,C-13 uniformly labeled proteins. The methods described are based on quantitative J correlation spectroscopy pioneered by Bar and co-workers [Bar et al. (1994) Methods Enzymol., 239, 79-105]. The combin...

Pulse sequences are presented for the measurement of3JC'C? and3JNC? scalar couplings for allC? containing residues in15N,13C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of 3JC'C? and3JNC? scalar...

The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side cha...

The near complete (>90%) NMR assignment of 15N, 13Cα, 13Cβ, and HN chemical shifts is presented for a 64 kDa trp repressor−operator complex consisting of two tandem dimers of 15N,13C,>90% 2H labeled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepressor, 5-methyltryptophan. The DNA sequence employed contains three copies of the palind...

The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals. Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which b...

Many structures of protein-protein complexes display large intermolecular interfaces. In a number of cases, however, mutagenesis and binding studies demonstrate that only a subset of the residues in the interfaces are responsible for the interaction energy. We have investigated the dynamic behavior of the C-terminal SH2 domain of phospholipase C an...

Protein-protein interfaces can consist of interactions between large numbers of residues of each molecule; some of these interactions are critical in determining binding affinity and conferring specificity, while others appear to play only a marginal role. Src-homology-2 (SH2) domains bind to proteins containing phosphorylated tyrosines, with addit...

A novel method is presented for the measurement of relaxation properties of methyl group deuterons in proteins uniformly C-13 labeled and fractionally deuterated. The experiments select for (CH2D)-C-13 methyl groups and make use of the excellent resolution provided by constant time C-13-H-1 correlation spectroscopy to measure the H-2 relaxation rat...

Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived...

A suite of NMR pulse schemes is presented for the assignment of HN, N-15, C-13(alpha), and C-13(beta) resonances in N-15, C-13, H-2 labeled proteins. The experiments exploit the line narrowing of the C-13 spins caused by the substitution of deuterons for bound protons and are therefore well suited for application to molecules with masses on the ord...

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradient...

The sensitivities of a number of gradient and nongradient versions of triple-resonance experiments are compared by quantitating the signal-to-noise ratios in spectra recorded on Cellulomonas fimi cellulose binding domain (110 amino acids), Xenopus laevis calmodulin (148 amino acids), Mycococcus xanthus protein S (173 amino acids), and a 93-amino ac...

New two-dimensional NMR experiments with high sensitivity and resolution are presented for the measurement of T-1, T-1 rho, and steady-state H-1-C-13 NOE values for CH C-13(alpha) spin systems in highly enriched, uniformly C-13-labeled proteins. Using a sample consisting of approximately equimolar amounts of 99% C-13(alpha)-alanine and 99% uniforml...

The interpretation of the resonance Raman (RR) data for chromophoric acyl serine proteases is rendered complex due to the fact that the almost-equal-to 350-nm laser beam used to generate the RR spectrum causes the substrate to photoisomerize in the active site. As a result, a steady-state population of dark (present before irradiation) and light (p...

An enhanced-sensitivity gradient 4D 15N, 13C-edited NOESY experiment is presented. Gradients are employed to suppress artifacts, eliminate the intense H2O signal and select for the coherence transfer pathway involving 15N magnetization. The latter use of the gradients results in a decrease in the number of phase cycle steps by a factor of two relat...

A J-compensated homonuclear spin-echo sequence is developed from the 90αo180α+2π/3o composite pulse using a product-operator analogy between homonuclear spin-spin coupling and RF pulses, and a J-compensated INADEQUATE sequence is constructed using this spin-echo sequence. Detailed study of the efficiencies of various composite pulses in minimizing...

Methods for the CHn editing of HOHAHA relay maps using DEPT and techniques for the suppression of directly bonded correlations have been developed. The complexity of HOHAHA relay maps can be reduced significantly and ambiguities due to overlap may be eliminated using these methods. A new broadband “purge” sequence was found to be most effective in...

A product operator description of the effects of chemical exchange in NMR experiments is developed. The utility of the product operator description is shown in an application to the DEPT magnetization transfer experiment. The results of DEPT experiments on N-acetylpyrrole (CH … CH) exchange) and N,N-dimethylacetamide (CH3 … CH3 exchange) showed tha...

Submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry. Thesis (Ph.D.)--University of Alberta, 1987.