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L E Kay

Publications and source records attributed to L E Kay.

At least 55 records · Page 3Linked to original sources
PubMed1998

A study of protein side-chain dynamics from new 2H auto-correlation and 13C cross-correlation NMR experiments: application to the N-terminal SH3 domain from drk.

Two new NMR experiments are presented for measuring side-chain dynamics in proteins. The first method, requiring 15N, 13C, approximately 50% 2H-labeled protein, measures 2H T1 and T1p spin relaxation times at side-chain positions. A second experiment permits the straightforward measurement of 13C-1H dipole-dipole cross-correlation relaxation rates at 13C beta positions in 15N, 13C-labeled molecules. An excellent correlation is observed between order parameters, describing the amplitude of motion at these sites, obtained on the basis of 2H relaxation and dipole-dipole cross-correlation relaxation rates. Together these experiments provide a powerful approach for selecting appropriate motional models. The methods are applied to study the side-chain motional properties of the N-terminal SH3 domain from the signaling protein drk.

Animals↗
PubMed1998

Analytical description of the effect of adiabatic pulses on IS, I2S, and I3S spin systems.

An analytical description of the evolution of magnetization in InS spin systems (1 </= n </= 3) during the course of an adiabatic pulse applied on spin S is provided. Calculations show that multiple-quantum terms are created during the pulse and that the rate at which in-phase and antiphase I-spin magnetization components interchange during spin-echo-based pulse sequences is decreased relative to the case where a hard inversion pulse is substituted for the adiabatic pulse. This has important consequences for purging schemes making use of such frequency-swept pulses. Simulations demonstrate that the evolution of in-phase I magnetization is essentially independent of n.

Magnetic Resonance Spectroscopy↗
PubMed1998

Independent ligand-induced folding of the RNA-binding domain and two functionally distinct antitermination regions in the phage lambda N protein.

The transcriptional antitermination protein N of bacteriophage lambda binds the boxB component of the RNA enhancer nut (boxA + boxB) and the E. coli elongation factor NusA. Efficient antitermination by N requires an RNA-binding domain (amino acids 1-22) and two activating regions for antitermination: a newly identified NusA-binding region (amino acids 34-47) that suppresses NusA's enhancement of termination, and a carboxy-terminal region (amino acids 73-107) that interacts directly with RNA polymerase. Heteronuclear magnetic resonance experiments demonstrate that N is a disordered protein. Interaction with boxB RNA induces only the RNA-binding domain of N to adopt a folded conformation, while the activating regions of the protein remain disordered in the absence of their target proteins.

Amino Acid Sequence↗
PubMed1998

Subunit-specific backbone NMR assignments of a 64 kDa trp repressor/DNA complex: a role for N-terminal residues in tandem binding.

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 resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound delta 2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.

Amino Acid Sequence↗
PubMed1998

Structure of a Numb PTB domain-peptide complex suggests a basis for diverse binding specificity.

The phosphotyrosine-binding (PTB) domain of Numb, a protein involved in asymmetric cell division, has recently been shown to bind to the adapter protein Lnx through an LDNPAY sequence, to the Numb-associated kinase (Nak) through a sequence that does not contain an NPXY motif and to GP(p)Y-containing peptides obtained from library screening. We show here that these diverse peptide sequences bind with comparable affinities to the Numb PTB domain at a common binding site on the surface of the protein. The NMR structure of the Numb PTB domain in complex with a GPpY-containing peptide reveals a novel mechanism of binding with the peptide in a helical turn that does not hydrogen bond to the PTB domain beta-sheet. These results suggest that PTB domains can potentially have multiple modes of peptide recognition and provide a structural basis from which the multiple functions of the Numb PTB domain during asymmetric cell division could arise.

Amino Acid Sequence↗
PubMed1998

Protein dynamics from NMR.

In the past several years a significant number of new multidimensional NMR methods have been developed to study molecular dynamics spanning a wide range of time scales. Applications involving a large number of biological systems have emerged and correlations with function established. Unique insights are obtained that are not available from structure alone, indicating the importance of dynamics studies for understanding function.

Binding Sites↗
PubMed1998

Correlation between binding and dynamics at SH2 domain interfaces.

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. Thus, structural data alone was not predictive of affinities in binding studies of phospholipase Cgamma1 and Syp phosphatase SH2 domains with phosphopeptides. NMR relaxation experiments probing dynamics of methyl groups of these complexes indicate a correlation between binding energy and restriction of motion at the interfacial region responsible for specific binding.

Insulin Receptor Substrate Proteins↗
PubMed1998

Protein dynamics from NMR.

The past several years have seen the development of a significant number of new multidimensional NMR methods for the study of molecular dynamics spanning a wide range of time scales. Applications involving a large number of different biological systems have emerged and correlations with function have been established. Unique insights are obtained that are not available from structure alone, indicating the importance of dynamics studies for understanding function.

Animals↗
PubMed1998

NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel.

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.

Amino Acid Sequence↗
PubMed1998

The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins.

During the past thirty years, deuterium labeling has been used to improve the resolution and sensitivity of protein NMR spectra used in a wide variety of applications. Most recently, the combination of triple resonance experiments and 2H, 13C, 15N labeled samples has been critical to the solution structure determination of several proteins with molecular weights on the order of 30 kDa. Here we review the developments in isotopic labeling strategies, NMR pulse sequences, and structure-determination protocols that have facilitated this advance and hold promise for future NMR-based structural studies of even larger systems. As well, we detail recent progress in the use of solution 2H NMR methods to probe the dynamics of protein sidechains.

Carbon Isotopes↗
PubMed1997

Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation.

The backbone dynamics of both folded and unfolded states of staphylococcal nuclease (SNase) and the N-terminal SH3 domain from drk (drkN SH3) are studied at two different temperatures. A simple method for obtaining order parameters, describing the amplitudes of motion of bond vectors, from NMR relaxation measurements of both folded and unfolded proteins is presented and the data obtained for 15N-NH bond vectors in both the SNase and drkN SH3 systems analyzed with this approach. Using a recently developed theory relating the amplitude of bond vector motions to conformational entropy, the entropy change between the folded and unfolded forms of SNase is calculated on a per residue basis. It is noteworthy that the region of the molecule with the smallest entropy change includes those residues showing native-like structure in the unfolded form of the molecule, as established by NOE-based experiments. Order parameters of backbone 15N-NH bond vectors show significantly larger changes with temperature in the unfolded states of both proteins relative to the corresponding folded forms. The differential temperature dependence is interpreted in terms of differences in the heat capacities of folded and unfolded polypeptide chains. The contribution to the heat capacity of the unfolded chain from rapid 15N-NH bond vector motions is calculated and compared with estimates of the heat capacity of the backbone unit, -CHCONH-, obtained from calorimetric data. Methyl dynamics measured at 14 and 30 degrees C establish that the amplitudes of side-chain motions in the folded SH3 domain are more sensitive to changes in temperature than the backbone dynamics, suggesting that over this temperature range side-chain ps to ns time-scale motions contribute more to the heat capacity than backbone motions for this protein.

Drosophila Proteins↗
PubMed1997

Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Delta131Delta, using NMR methods with improved resolution.

Comprehensive NOE results from detailed structural characterization of a 131 residue partially folded fragment of staphylococcal nuclease (Delta131Delta) made possible by NMR methods with improved resolution are presented. The resulting NOE patterns reflect sampling of both alpha and beta regions of phi, phi conformational space, yet demonstrate significant preferences for both native-like and non-native-like turn and potentially helical conformations. Together with data from studies of the unfolded state of the drkN SH3 domain, NOE patterns observed for partially folded or unfolded proteins are summarized. It is surprising that few long-range NOEs were observed in Delta131Delta. The two longest-range NOEs are both native-like; one of these, an (i,i+5) NOE, provides evidence for a Schellman capping motif for helix termination. Many aliphatic-aliphatic and aliphatic-amide NOEs, which are not normally observed in folded proteins, were detected. We have ruled out significant contributions from spin-diffusion for a number of these NOEs and suggest that one source may be sampling of non-prolyl cis peptide bond configurations in the disordered state of Delta131Delta.

Amino Acid Sequence↗
PubMed1997

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif.

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYphi motif (phi represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 +/- 0.74 microM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 +/- 0.10 microM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

Animals↗
PubMed1997

Solution structure of recombinant human interleukin-6.

Interleukin-6 (IL-6) is a 185 amino acid cytokine which exerts multiple biological effects in vivo and whose dysregulation underlies several disease processes. The solution structure of recombinant human interleukin-6 has now been determined using heteronuclear three and four-dimensional NMR spectroscopy. The structure of the molecule was determined using 3044 distance and torsion restraints derived by NMR spectroscopy to generate an ensemble of 32 structures using a combined distance geometry/simulated annealing protocol. The protein contains five alpha-helices interspersed with variable-length loops; four of these helices constitute a classical four-helix bundle with the fifth helix located in the CD loop. There were no distance violations greater than 0.3 A in any of the final 32 structures and the ensemble has an average-to-the-mean backbone root-mean-square deviation of 0.50 A for the core four-helix bundle. Although the amino-terminal 19 amino acids are disordered in solution, the remainder of the molecule has a well defined structure that shares many features displayed by other long-chain four-helix bundle cytokines. The high-resolution NMR structure of hIL-6 is used to rationalize available mutagenesis data in terms of a heteromeric receptor complex.

Antigens, CD↗
PubMed1997

Characterization of the backbone dynamics of folded and denatured states of an SH3 domain.

Measurements of 15N NMR relaxation parameters have been used to characterize the backbone dynamics of folded and denatured states of the N-terminal SH3 domain from the adapter protein drk, in high salt or guanidinium chloride, respectively. Values of the spectral density function evaluated at a number of frequencies are compared. The levels of backbone dynamics in the folded protein show little variation across the molecule and are of similar magnitude to those determined previously for the folded state of the protein in exchange with an unfolded state at low salt concentrations [Farrow et al. (1995) Biochemistry 34, 868-878]. The denatured state of the domain exhibits both more extensive and more heterogeneous dynamics than the folded state. In particular the profile of the spectral density function evaluated at zero-frequency for the unfolded state of the domain indicates that residues in the middle of the protein sequence are considerably less mobile than those at the termini. These data suggest that the molecule is not behaving as an extended polymer and that concerted motions of the central portions of the molecule are occurring, consistent with a reasonably compact conformation in this region. The backbone dynamics of the folded and unfolded states were studied at two temperatures. The level of high-frequency motions in the folded molecule is largely unaffected by changes in temperature, whereas an increase in temperature results in increased high-frequency motion in the unfolded state.

Animals↗
PubMed1997

Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR.

The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deuterons significantly improves the sensitivity of many of the experiments used in chemical shift assignment. Unfortunately, uniform deuteration also radically depletes the number of interproton distance restraints available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterated proteins through the use of deuterated, selectively methyl-protonated samples. This labeling profile maintains the efficiency of triple-resonance NMR experiments while retaining a sufficient number of protons at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how this deuteration scheme affects the sensitivity and resolution of experiments used to assign 15N, 13C, and 1H chemical shifts and interproton NOEs. This approach is tested experimentally on a 14 kDa SH2/phosphopeptide complex, and a global protein fold is obtained from a set of methyl-methyl, methyl-NH, and NH-NH distance restraints. We demonstrate that the inclusion of methyl-NH and methyl-methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH-NH distance restraints. Finally, we examine the general applicability of this approach by determining the structures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.

Deuterium↗