Griselda Hernández
New York State Department of Health
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Featured researches published by Griselda Hernández.
Proteins | 2005
David M. LeMaster; Jianzhong Tang; Diana I. Paredes; Griselda Hernández
The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi‐turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi‐turn sequence into the mesophile protein sequence yields a hybrid, (14–33Pf) Cp, that exhibits a 12° increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer‐based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14–33Pf) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi‐turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14–33Pf) Cp hybrid, similar to that observed for the multi‐turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs. Proteins 2005.
Biochemistry | 2009
Griselda Hernández; Janet S. Anderson; David M. LeMaster
Hydroxide-catalyzed exchange rate constants were determined for those amides of FK506-binding protein (FKBP12), ubiquitin, and chymotrypsin inhibitor 2 (CI2) that are solvent-accessible in the high-resolution X-ray structures. When combined with previous hydrogen exchange results for the rubredoxin from Pyrococcus furiosus, the acidity of these amides was calculated by continuum dielectric methods as a function of the nonpolarizable electrostatic parameter set, internal dielectric, and the charge distribution of the peptide anion. The CHARMM22 parameter set with an internal dielectric value of 3 and an ab initio-derived anion charge distribution yielded an rmsd value of 7 for the 56 amide exchange rate constants ranging from 10(0.67) to 10(9.0) M(-1) s(-1). The OPLS-AA parameter set yielded comparably robust predictions, while that of PARSE, AMBER parm99, and AMBER ff03 performed more poorly. The small value for the optimal internal dielectric, combined with the brief lifetime of the peptide anion intermediate and the uniformity of the correlation between predicted and observed amide acidities, is consistent with electronic polarizability providing the dominant contribution to dielectric shielding. By construction, nonpolarizable force fields do not model electric field attenuation by electronic polarizability. Accurate prediction of the total electrostatic energy by such force fields necessitates the hyperpolarization of the atomic charge values in order to match the average electric field energy density (1/2)epsilon(tau)E(2)(tau) when epsilon(tau) is set to the in vacuo dielectric value of 1. The resulting predictions of the experimental hydrogen exchange data demonstrate the substantial systematic errors in the predicted electrostatic potential that can arise when dielectric shielding due to electronic polarizability is neglected.
Biochemistry | 2009
David M. LeMaster; Janet S. Anderson; Griselda Hernández
The amide hydrogens that are exposed to solvent in the high-resolution X-ray structures of ubiquitin, FK506-binding protein, chymotrypsin inhibitor 2, and rubredoxin span a billion-fold range in hydroxide-catalyzed exchange rates which are predictable by continuum dielectric methods. To facilitate analysis of transiently accessible amides, the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin were determined under near physiological conditions. With the previously reported NMR-restrained molecular dynamics ensembles of ubiquitin (PDB codes 2NR2 and 2K39) used as representations of the Boltzmann-weighted conformational distribution, nearly all of the exchange rates for the highly exposed amides were more accurately predicted than by use of the high-resolution X-ray structure. More strikingly, predictions for the amide hydrogens of the NMR relaxation-restrained ensemble that become exposed to solvent in more than one but less than half of the 144 protein conformations in this ensemble were almost as accurate. In marked contrast, the exchange rates for many of the analogous amides in the residual dipolar coupling-restrained ubiquitin ensemble are substantially overestimated, as was particularly evident for the Ile 44 to Lys 48 segment which constitutes the primary interaction site for the proteasome targeting enzymes involved in polyubiquitylation. For both ensembles, “excited state” conformers in this active site region having markedly elevated peptide acidities are represented at a population level that is 102 to 103 above what can exist in the Boltzmann distribution of protein conformations. These results indicate how a chemically consistent interpretation of amide hydrogen exchange can provide insight into both the population and the detailed structure of transient protein conformations.
ChemBioChem | 2008
Griselda Hernández; Janet S. Anderson; David M. LeMaster
The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the α2 helix. Amides in the active site exhibit diffusion‐limited, hydroxide‐catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide‐catalyzed exchange by a million‐fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition‐state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.
Proteins | 2005
Griselda Hernández; David M. LeMaster
Given any operational criterion for pairwise interatomic interactions, for a pair of structurally homologous proteins there exists for both proteins a unique equivalent partitioning of the nonconserved residue positions into mutually non‐interacting clusters. In the formation of a chimeric protein derived from these two parental sequences, if nonnative‐like interactions are to be avoided in its tertiary structure, then all of the nonconserved residues of each cluster must necessarily be either maintained or interchanged simultaneously. This hybrid native partitioning criterion is applied to known gene shuffling results. When the degree of estimated disruption is modest, the HybNat algorithm provides an efficient predictor of structural integrity. This supports the expectation that a substantial fraction of sequences that conform to the hybrid native partitioning criterion will yield tertiary structures that largely preserve the native‐like interactions of the parental proteins. Proteins 2005.
Proteins | 2004
David M. LeMaster; Jianzhong Tang; Griselda Hernández
The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability. This in turn implies a corresponding retardation of the folding reaction. A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100°C of nearly a week. Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144°C (137°C for the N‐terminal modified A2K variant) and 104°C, respectively, with similar (un)folding rates of ≈25,000 s−1, only modestly slower than the diffusion controlled rate. The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability. The two‐state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (Tm) of nearly 200°C. This discrepancy is resolved by the observation that the base‐catalyzed hydrogen exchange of the model dipeptide (N‐acetyl‐L‐cysteine‐N‐methylamide)4‐Cd2+ is 23‐fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data. Proteins 2004.
Biophysical Chemistry | 2009
Janet S. Anderson; Griselda Hernández; David M. LeMaster
Electrostatic interactions at the protein surface yield over a billion-fold range of amide hydrogen exchange rates. This range is equivalent to the maximal degree of attenuation in exchange rates that have been shown to occur for amides buried within the protein interior. Continuum dielectric analysis of Ala-Ala, Ala-Gly, Gly-Ala and trans-Pro-Ala peptide conformer acidities predicts that the relative orientation of the two neighboring peptide groups can account for a million-fold variation in hydroxide-catalyzed hydrogen exchange rates. As in previous protein studies, an internal dielectric value of 3 was found to be applicable to simple model peptides, presumably reflecting the short lifetime of the peptide anion intermediate. Despite the million-fold range in conformer acidities, the small differences in the experimental exchange rates for these peptides are accurately predicted. Ala-Ala conformers with an extended N-terminal residue and the C-terminal residue in the alpha conformation are predicted to account for over 60% of the overall hydrogen exchange reaction, despite constituting only 12% of the protein coil population.
Biochemical Journal | 2013
Sourajit M. Mustafi; Hui Chen; Hongmin Li; David M. LeMaster; Griselda Hernández
The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80′s loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the β-sheet, across the active site to the α-helix and into the 50′s loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80′s loop. Line-broadening was also decreased for the residues in the α-helix and 50′s loop, whereas line-broadening in the 40′s loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40′s loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.
Biochemical Journal | 2014
Sourajit M. Mustafi; David M. LeMaster; Griselda Hernández
As co-chaperones of Hsp90 (heat-shock protein 90), FKBP51 (FK506-binding protein of 51 kDa) and FKBP52 (FK506-binding protein of 52 kDa) act as antagonists in regulating the hormone affinity and nuclear transport of steroid receptor complexes. Exchange of Leu119 in FKBP51 for Pro119 in FKBP52 has been shown to largely reverse the steroid receptor activities of FKBP51 and FKBP52. To examine whether differences in conformational dynamics/plasticity might correlate with changes in the reported receptor activities, 15N-NMR relaxation measurements were carried out on the N-terminal FKBP domains of FKBP51 and FKBP52 as well as their residue-swapped variants. Both proteins exhibit a similar pattern of motion in the picosecond–nanosecond timeframe as well as a small degree of 15N line-broadening, indicative of motion in the microsecond–millisecond timeframe, in the β3a strand of the central sheet. Only the FKBP51 domain exhibits much larger line-broadening in the adjacent β3 bulge (40′s loop of FKBP12) and throughout the long β4–β5 loop (80′s loop of FKBP12). The L119P mutation at the tip of the β4–β5 loop completely suppressed the line-broadening in this loop while partially suppressing the line-broadening in the neighbouring β2 and β3a strands. The complementary P119L and P119L/P124S variants of FKBP52 yielded similar patterns of line-broadening for the β4–β5 loop as that for FKBP51, although only 20% and 60% as intense respectively. However, despite the close structural similarity in the packing interactions between the β4–β5 loop and the β3a strand for FKBP51 and FKBP52, the line-broadening in the β3a strand is unaffected by the P119L or P119L/P124S mutations in FKBP52.
Journal of Biological Chemistry | 2015
David M. LeMaster; Sourajit M. Mustafi; Matthew Brecher; Jing Zhang; Annie Heroux; Hongmin Li; Griselda Hernández
Background: FK506-binding protein 51 (FKBP51) inhibits and FKBP52 stimulates transcription by various steroid receptors. Exchange of a single residue largely reverses this pattern of regulation. Results: Unlike FKBP52, FKBP51 has two distinct coupled conformational transitions surrounding this mutation site. Conclusion: Structural analysis of FKBP51 transient states can inform inhibitor design by conformational selection. Significance: The differential plasticity of the FKBP domains may underlie their differential regulation. Interchanging Leu-119 for Pro-119 at the tip of the β4-β5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β4-β5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β2 and β3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β2 and β3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.