You are here
Griselda Hernández, Ph.D.
Griselda Hernández, Ph.D.
- Nuclear Magnetic Resonance Structural Biology
- Adjunct Assistant Professor, School of Public Health, Biomedical Sciences
- Ph.D., University of Rochester (1991)
- Postdoctoral training: University of California at Davis
- Postdoctoral training: Los Alamos National Laboratory
firstname.lastname@example.org (518) 474-4673 Fax: (518) 473-2900
We study the flexibility of protein molecules in solution as it relates to intramolecular signaling in allosteric interactions and the factors controlling thermostability among homologous proteins that have evolved at differing temperatures. Understanding of these problems is of current interest in the design of drug binding and stability of protein based therapies.
The earliest direct evidence for the dynamic character of protein conformations came from measurements demonstrating a very wide range of rates by which the backbone amide hydrogens of a protein exchange with bulk solvent. Traditionally these data have been interpreted assuming that the degree to which these exchange rates are slower than those of simple model peptides provides a direct prediction of the fraction of time that an amide hydrogen is exposed to solvent. However, the kinetic acidity of the amide hydrogen has long been known to be directly determined by its thermodynamic acidity, which in turn is highly sensitive to the local electrostatic environment. As a result, peptide hydrogen exchange that occurs from a protein conformation which retains any tertiary structure can be expected to reflect the intramolecular electrostatic interactions within that conformation. We determined that the hydroxide-catalyzed exchange rate constants for the backbone amides that are exposed to solvent in several small proteins span a billion-fold range. Furthermore, we can predict these rates with reasonable accuracy by continuum dielectric methods.
Protein flexibility is fundamentally characterized by the Boltzmann-weighted population of protein conformations. Particularly regarding weakly populated states, accurate characterization by either theoretical modeling or experimental measurement remains problematic. To analyze such transiently accessible amides, we have measured the hydroxide-catalyzed rate constants for every backbone amide of ubiquitin under near physiological conditions. We have carried out continuum dielectric predictions of the backbone amide acidities using structures from previously reported molecular dynamics ensembles of ubiquitin. For both the NMR relaxation-restrained 2NR2 ensemble and the NMR residual dipolar coupling-restrained 2K39 ensemble, 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. Furthermore, for the 2NR2 ensemble the amide hydrogens that become exposed to solvent in more than one, but less than half of the 144 protein conformations were predicted almost as accurately. In marked contrast, the exchange rates for many of the analogous amides in the 2K39 ensemble are substantially overestimated, indicating that this modeled population strongly deviates from the correct Boltzmann-weighted conformational distribution. 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.
We have designed complementary hybrids derived from the mesophile and hyperthermophile homologous rubredoxins Clostridium pasteurianum and Pyrococcus furiosus endowed with additivity in their structure, dynamics and thermal stability. We are currently applying these hybrid design principles to other pairs of evolutionarily related proteins. Perfectly conserved interface hybrids can enable delineation of the spatial distribution of dynamics and quantification of the contribution to thermal stability from the interchanged segments thus providing an excellent playground for computational analysis of conformational flexibility.