A5043188408- Protein Structure and Dynamics
- Enzyme Structure and Function
- RNA and protein synthesis mechanisms
- Ubiquitin and proteasome pathways
- Biochemical and Molecular Research
- RNA modifications and cancer
- Hemoglobin structure and function
- RNA Research and Splicing
- DNA and Nucleic Acid Chemistry
- Connective tissue disorders research
- Advanced Topology and Set Theory
- Porphyrin Metabolism and Disorders
- Heat shock proteins research
- HIV/AIDS drug development and treatment
- Cancer-related gene regulation
- HIV Research and Treatment
- Insect symbiosis and bacterial influences
- Legume Nitrogen Fixing Symbiosis
- Studies on Chitinases and Chitosanases
- Fuzzy and Soft Set Theory
University of California, San Francisco
2015-2022
University of Pittsburgh
2009-2013
Rational design and directed evolution have proved to be successful approaches increase catalytic efficiencies of both natural artificial enzymes. Protein dynamics is recognized as important, but due the inherent flexibility biological macromolecules it often difficult distinguish which conformational changes are directly related function. Here, we use on an impaired mutant proline isomerase CypA identify two second-shell mutations that partially restore its activity. We show kinetically,...
Abstract The creation of artificial enzymes is a key objective computational protein design. Although de novo have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography study changes in the conformational ensemble during designed Kemp eliminase HG3 ( k cat / K M 146 −1 s ). We observe that residues are increasingly rigidified, active site becomes better pre-organized, and its...
Naturally occurring proteins vary the precise geometries of structural elements to create distinct shapes optimal for function. We present a computational design method, loop-helix-loop unit combinatorial sampling (LUCS), that mimics nature's ability families with same overall fold but precisely tunable geometries. Through near-exhaustive elements, LUCS generates highly diverse encompassing those found in nature also surpassing known structure space. Biophysical characterization showed 17...
Domain swapping creates protein oligomers by exchange of structural units between identical monomers. At present, no unifying molecular mechanism domain has emerged. Here we used the Cyanovirin-N (CV-N) and (19)F-NMR to investigate process swapping. CV-N is an HIV inactivating that can exist as a monomer or domain-swapped dimer. We measured thermodynamic kinetic parameters conversion determined size energy barrier two species. The very large similar magnitude for equilibrium unfolding...
During coevolution with the host, HIV-1 developed ability to hijack cellular ubiquitin/proteasome degradation pathway counteract antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block replication. Abrogation A3G function involves Vif protein, which binds and serves as an adapter molecule recruit Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis focused on 14 most surface-exposed Lys residues allowed us identify four (Lys-297, 301, 303, 334) are...
Significance The structural details of protein motions are critical to understanding many biological processes, but they often hidden conventional biophysical techniques. Diffuse X-ray scattering can reveal the correlated movements between atoms; however, data collection historically has required extra effort and dedicated experimental protocols. We have measured 3D diffuse intensities in diffraction from CypA trypsin crystals using standard crystallographic Analysis resulting is consistent...
Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, primary enzyme involved in degradation environmental lungs. Mutations to acidic chitinase have been associated with asthma, genetic deletion mice increases morbidity mortality age. We initially set out reverse this...
Although it has long been established that the amino acid sequence encodes fold of a protein, how individual proteins arrive at their final conformation is still difficult to predict, especially for oligomeric structures. Here, we present comprehensive characterization species cyanovirin-N all are formed by polypeptide chain with identical sequence. Structures oligomers were determined X-ray crystallography, and each one exhibits 3D domain swapping. One unique domain-swapped structure...
A detailed analysis of high-resolution structural data and computationally predicted dynamics was carried out for a designed sugar-binding protein. The mean-square deviations in the positions residues derived from nuclear magnetic resonance (NMR) models those inferred X-ray crystallographic B-factors two different crystal forms were compared with predictions based on Gaussian Network Model (GNM) results molecular (MD) simulations. GNM systematically yielded higher correlation than MD,...
SignificanceComputational protein design promises to advance applications in medicine and biotechnology by creating proteins with many new useful functions. However, functions require the of specific often irregular atom-level geometries, which remains a major challenge. Here, we develop computational methods that predict local geometries greater accuracy than existing methods. Then, as proof concept, leverage these conformations enzyme ketosteroid isomerase change protein's preference for...
Recent studies suggest that protein motions observed in molecular simulations are related to biochemical activities, although the computed time scales do not necessarily match those of experimentally processes. The origin this conflicting observation is explored here for a test protein, cyanovirin-N (CV-N), through series dynamics span range three orders magnitude up 0.4 μs. Strikingly, increasing simulation leads an approximately uniform amplification motional sizes, while maintaining same...
Abstract X-ray diffraction has the potential to provide rich information about structural dynamics of macromolecules. To realize this potential, both Bragg scattering, which is currently used derive macromolecular structures, and diffuse reports on correlations in charge density variations must be measured. Until now measurement scattering from protein crystals been scarce, due extra effort collecting data. Here, we present three-dimensional measurements intensity collected enzymes...