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J D Forman-Kay

Publications and source records attributed to J D Forman-Kay.

At least 37 records · Page 2Linked to original sources
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

NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions.

The isolated N-terminal SH3 domain of the Drosophila adapter protein drk (drkN SH3 domain) exists in a dynamic equilibrium between a folded (F(exch)) and an unfolded (U(exch)) state under native-like buffer conditions [Zhang, O., & Forman-Kay, J. D. (1995) Biochemistry 34, 6784-6794]. The effect of binding a proline-rich peptide derived from the protein Sos, a biological target of the drkN SH3 domain, on this equilibrium has been investigated. The stabilization of the F(exch) folded state upon binding provides an example of the link between binding and protein folding or stabilization. We have compared NMR parameters of the U(exch) state with those of a denatured state in 2 M guanidine hydrochloride (U(Gdn)). Variable-temperature experiments demonstrate that interactions in a region encompassing residues Gln 23-Leu 28 in the U(exch) state are destabilized upon addition of guanidine hydrochloride. Data from an 15N HSQC-NOESY-HSQC experiment as well as recently developed methods provide more unambiguous structural information than described previously, showing the presence of preferential structure in both unfolded states. Backbone NOEs observed in both unfolded states as well as chemical shifts and coupling constants suggest a rapid equilibrium between extended structure and turn-like structures which may play a role in initiation of protein folding. However, differences in detailed structural features between the two unfolded states argue that caution is needed in interpretation of results from structural characterization of protein conformational states generated using denaturing conditions.

Amino Acid Sequence↗
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

pH titration studies of an SH2 domain-phosphopeptide complex: unusual histidine and phosphate pKa values.

Electrostatic interactions in a complex of the phospholipase C-gamma 1 C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the beta platelet-derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His beta D4) and the phosphotyrosine (pTyr) phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pKa of His beta D4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein.

Amides↗
PubMed1997

Triple-resonance NOESY-based experiments with improved spectral resolution: applications to structural characterization of unfolded, partially folded and folded proteins.

NMR-based structural studies of macromolecules focus to a large extent on the establishment of interproton distances within the molecule based on the nuclear Overhauser effect (NOE). Despite the improvements in resolution resulting from multidimensional NMR experiments, the detailed characterization of disordered states of proteins or highly overlapped regions of folded molecules using current NMR methods remains challenging. A suite of triple-resonance NOESY-type pulse schemes is presented which require uniform 15N and 13C labeling and make use of the chemical shift dispersion of backbone 15N and 13C' (carbonyl) resonances to increase the spectral resolution. In particular, for the case of partially folded and unfolded proteins, the experiments exploit the fact that the dispersion of 15N and 13C' resonances is comparable to that observed in folded states. Ambiguities that arise in the assignment of NOEs as a result of the severe chemical shift degeneracy in 1H and aliphatic 13C nuclei are resolved, therefore, by recording the chemical shifts of 15N or 13C' either before or after the NOE mixing period. Applications of these methods to the study of the unfolded state of the N-terminal SH3 domain of drk (drkN SH3) and a partially folded large fragment of staphylococcal nuclease (SNase), delta 131 delta, are presented. In addition, an application to folded SNase in complex with the ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+ is illustrated which allows the assignment of NOEs between degenerate H alpha protons or protons resonating close to water.

Amino Acid Sequence↗
PubMed1996

Correlation between dynamics and high affinity binding in an SH2 domain interaction.

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 additional specificity provided by interactions with residues C-terminal to the phosphotyrosine (pTyr) residue. While the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2) interacts with eight residues of a pTyr-containing peptide from its high affinity binding site on the beta-platelet-derived growth factor receptor, it can still bind tightly to a phosphopeptide containing only three residues. Novel deuterium (2H) based nuclear magnetic resonance (NMR) spin relaxation experiments which probe the nanosecond-picosecond time scale dynamics of methyl containing side chain residues have established that certain regions of the PLCC SH2 domain contacting the residues C-terminal to the pTyr have a high degree of mobility in both the free and peptide complexed states. In contrast, there is significant restriction of motion in the pTyr binding site. These results suggest a correlation between the dynamic behavior of certain groups in the PLCC SH2 complex and their contribution to high affinity binding and binding specificity.

Amino Acid Sequence↗
PubMed1995

Structural and dynamic characterization of the phosphotyrosine binding region of a Src homology 2 domain--phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches.

Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pocket of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and possibly the Arg 18 guanidino groups.

Amino Acid Sequence↗
PubMed1995

Structural characterization of folded and unfolded states of an SH3 domain in equilibrium in aqueous buffer.

The isolated N-terminal Src homology 3 (SH3) domain of Drosophila drk exists in equilibrium between folded and unfolded states in aqueous buffer near neutral pH. Nuclear magnetic resonance spectra recorded on both states simultaneously exhibit an approximate 1:1 ratio of protein conformations. The folded form is similar to other known SH3 structures, especially the N-terminal SH3 domain of the mammalian homologue GRB2. A stretch of sequential amide-amide nuclear Overhauser effect cross-peaks for resonances of the unfolded state is observed in a region corresponding to beta-strands in the folded state. The results suggest that turn-like conformations may be preferentially sampled in the folding pathway for this predominantly beta-structured SH3 domain. In addition, a stable turn at Leu-28 is observed in the unfolded but not in the folded state. Comparison of this unfolded form with a denatured state in 2 M guanidine hydrochloride shows that, while both are highly disordered, these states are not identical and more residual structure is present under nondenaturing conditions.

Amino Acid Sequence↗
PubMed1995

Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer.

Two-dimensional NMR 15N relaxation studies have been used to characterize the backbone dynamics and folding transition of the N-terminal SH3 domain of the protein drk (drkN SH3). The isolated drkN SH3 domain exists in equilibrium between a folded and an unfolded state in aqueous buffer and near neutral pH [Zhang et al. (1994) J. Biomol. NMR 4, 845]. The backbone dynamics of both the folded and unfolded states in this exchanging system have been determined and the rates of the folding transition estimated at 14 degrees C. For 12 residues, the values of the spectral density functions of the backbone amide bond vectors at a number of frequencies have been established. Results show that while the unfolded state has considerably greater dynamic behavior, the overall motional properties are consistent with it having a reasonably compact structure in solution. In contrast to the folded state, considerable variations are seen in the values of the spectral densities of the unfolded state as a function of residue number; these variations do not appear to be strongly correlated with structural elements in the folded state. The mean value of the exchange rate associated with the folding transition was determined to be 0.89 s-1, similar to previous measurements of the rate of formation of beta-structure.

Amino Acid Sequence↗
PubMed1994

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

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 gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Amino Acid Sequence↗
PubMed1994

Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide.

The solution structure of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLC-gamma 1), in complex with a phosphopeptide corresponding to its Tyr-1021 high affinity binding site on the platelet-derived growth factor receptor, has been determined by nuclear magnetic resonance spectroscopy. The topology of the SH2-phosphopeptide complex is similar to previously reported Src and Lck SH2 complexes. However, the binding site for residues C-terminal to the phosphotyrosine (pTyr) is an extended groove that contacts peptide residues at the +1 to +6 positions relative to the pTyr. This striking difference from Src and Lck reflects the fact that the PLC-gamma 1 complex involves binding of a phosphopeptide with predominantly hydrophobic residues C-terminal to the pTyr and therefore serves as a prototype for a second class of SH2-phosphopeptide interactions.

Amino Acid Sequence↗
PubMed1994

Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques.

The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.

Amino Acid Sequence↗
PubMed1994

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium.

A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, an approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

Chemical Phenomena↗
PubMed1992

Relationship between electrostatics and redox function in human thioredoxin: characterization of pH titration shifts using two-dimensional homo- and heteronuclear NMR.

The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) 1H and 15N nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear 1H-1H Hartmann-Hahn (HOHAHA) and heteronuclear 1H-15N Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pKas for certain isolated ionizable groups, including the single histidine residue at position 43 (pKa = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pKas for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (1991) Biochemistry 30, 4262-4268]. The pKas of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pKas are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pKas within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S gamma of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].

Binding Sites↗
PubMed1992

1H and 15N resonance assignments and secondary structure of the human thioredoxin C62A, C69A, C73A mutant.

The complete assignment of 1H and 15N backbone resonances and near-complete 1H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional 15N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay, J.D. et al. (1991) Biochemistry, 30, 2685-2698]. An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.

Amino Acid Sequence↗
PubMed1991

Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy.

The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy. It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules. Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules. Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions. Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.

Amino Acid Sequence↗
PubMed1991

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy.

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.

Amino Acid Sequence↗