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1.  Thermodynamic Stability of Histone H3 Is a Necessary but not Sufficient Driving Force for its Evolutionary Conservation 
PLoS Computational Biology  2011;7(1):e1001042.
Determining the forces that conserve amino acid positions in proteins across species is a fundamental pursuit of molecular evolution. Evolutionary conservation is driven by either a protein's function or its thermodynamic stability. Highly conserved histone proteins offer a platform to evaluate these driving forces. While the conservation of histone H3 and H4 “tail” domains and surface residues are driven by functional importance, the driving force behind the conservation of buried histone residues has not been examined. Using a computational approach, we determined the thermodynamically preferred amino acids at each buried position in H3 and H4. In agreement with what is normally observed in proteins, we find a significant correlation between thermodynamic stability and evolutionary conservation in the buried residues in H4. In striking contrast, we find that thermodynamic stability of buried H3 residues does not correlate with evolutionary conservation. Given that these H3 residues are not post-translationally modified and only regulate H3-H3 and H3-H4 stabilizing interactions, our data imply an unknown function responsible for driving conservation of these buried H3 residues.
Author Summary
Most proteins fold to a well-defined, three-dimensional structure, which can be delineated into the protein surface and its buried core. When comparing amino acid sequences of the same protein from different organisms, we would expect to find certain residue positions conserved due to the importance of that position in either maintaining the protein's function or its three-dimensional structure. In this study, we looked at residues in the buried core domains of histone proteins H3 and H4, which have no known function other than maintaining the three-dimensional structure of the protein. We find that perturbing protein stability (which is a measure of maintenance of the protein's structure) by mutating these residues compromises survival fitness in yeast. However, the stability conferred by buried amino acids of H3 alone cannot account for their evolutionary conservation, which is in striking contrast to other proteins where stability has been shown to be the driving force for sequence conservation. This conservation of H3 thus points to either new additional functions of H3 that have not been uncovered or a unique conservation mechanism that goes beyond survival pressure. These data therefore reveal a highly conserved domain that is distinct in its evolutionary conservation.
doi:10.1371/journal.pcbi.1001042
PMCID: PMC3017104  PMID: 21253558
2.  Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding 
PLoS Computational Biology  2008;4(2):e1000008.
The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant.
Author Summary
Deletion of a single residue, phenylalanine at position 508, in the first nucleotide binding domain (NBD1) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is present in approximately 90% of cystic fibrosis (CF) patients. Experiments show that this mutant protein exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant incorrect interactions of other domains. However, little is known about the direct effect of the Phe508 deletion on NBD1 folding. Here, using molecular dynamics simulations of NBD1-WT, NBD1-F508A, and NBD1-ΔF508, we show that the deletion of Phe508 indeed alters the kinetics of NBD1 folding. We also find that the intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. Moreover, we identified critical interactions not necessarily localized near position 508, such as Q493/P574 and F575/F587, to be significant structural elements influencing the kinetic difference between wild type and mutant NBD1. We propose that these observed alterations in folding kinetics of mutant NBD1 result in misassembly of the whole multi-domain protein, thereby causing its premature degradation.
doi:10.1371/journal.pcbi.1000008
PMCID: PMC2265529  PMID: 18463704
3.  Identification and Rational Redesign of Peptide Ligands to CRIP1, A Novel Biomarker for Cancers 
PLoS Computational Biology  2008;4(8):e1000138.
Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of cancers. Here we report on the use of phage display in combination with molecular modeling to identify a high-affinity ligand for CRIP1. Panning experiments using a circularized C7C phage library yielded several consensus sequences with modest binding affinities to purified CRIP1. Two sequence motifs, A1 and B5, having the highest affinities for CRIP1, were chosen for further study. With peptide structure information and the NMR structure of CRIP1, the higher-affinity A1 peptide was computationally redesigned, yielding a novel peptide, A1M, whose affinity was predicted to be much improved. Synthesis of the peptide and saturation and competitive binding studies demonstrated approximately a 10–28-fold improvement in the affinity of A1M compared to that of either A1 or B5 peptide. These techniques have broad application to the design of novel ligand peptides.
Author Summary
Breast cancer is one of the most frequently diagnosed malignancies in American females and is the second leading cause of cancer deaths in women. Several improvements in diagnostic protocols have enhanced our ability for earlier detection of breast cancer, resulting in improvement of therapeutic outcome and an increased survival rate for breast cancer patients. However, current early screening techniques are neither comprehensive nor infallible. Imaging techniques that improve breast cancer detection, localization, and evaluation of therapy are essential in combating the disease. Cysteine-rich intestinal protein 1 (CRIP1) has been identified as a novel marker for early detection of breast cancers. Here, we report the use of phage display and computational molecular modeling to identify a high-affinity ligand for CRIP1. Phage display panning experiments initially identified consensus peptide sequences with modest binding affinity to purified CRIP1. Using ab initio modeling of binding peptide structures, computational docking, and recently developed free energy estimation protocols, we redesigned the peptides to increase their affinity for CRIP1. Synthesis of the redesigned peptide and binding studies demonstrated approximately a 10–28-fold improvement in the binding affinity. The combination of computational and experimental techniques in this study demonstrates a potentially powerful tool in modulating protein–protein interactions.
doi:10.1371/journal.pcbi.1000138
PMCID: PMC2453235  PMID: 18670594

Results 1-3 (3)