HYBRID PROTEIN ENGINEERING

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Designing a protein sequence which folds into a desired three-dimensional shape is known as the inverse protein-folding problem. In nature, protein sequences are limited to combinations of the naturally occurring 20 amino acids and their post-translational modifications. Incorporation of non-natural amino acids provides unique possibilities for designing proteins that adopt a stable fold. For example, incorporation of Aminoisobutyric acid (Aib) into proteins restricts the phi and psi torsion angles of that residue and is known to promote helix formation. It is believed that Aib lowers the entropic penalty of helix formation upon protein folding through preorganization. By the same principle, incorporating semi-rigid mimetics of a-helices, b-sheets, and reverse-turns into a protein would minimize the entropy loss on folding through preorganization while retaining the interactive surface features that optimize the favorable enthalpic interactions in the folded state. A semi-rigid mimetic reduces a proteins fold space and should promote protein folding by nucleation. Modular secondary structure mimetics can serve as building blocks in the design of ultra-stable, catalytically active proteins that resist degradation. BBA foldamers: The 28-residue FSD-1 betabeatalpha (BBA) protein of Dahiyat and Mayo was computationally designed to form a stable zinc-finger BBA fold independent of zinc binding. The 12 residue alpha-helical region of FSD-1 will be replaced with a designed helix mimetic to form a hybrid protein that is part natural peptide and part peptidomimetic. This hybrid protein is expected to be more stable than FSD-1 because the entropic penalty upon folding is reduced. Molecular dynamics simulations in explicit water using GROMACS 3.3 can be performed on a long (50-100 nanoseconds) time scale to predict its stability. A hybrid semi-synthetic protein containing a ligated helical peptidomimetic aimed at further stabilizing the BBA fold has been designed. Ribonuclease A - RNAse is an enzyme which can be cleaved into S-peptide (residues 1-20) and S-protein (residues 21-124) by subtilisin. Neither S-peptide nor S-protein is enzymatically active on its own. Re-introducing a shortened S-peptide (residues 1-14) into a solution with S-protein at 1:1 molar ratio restores full enzymatic activitythis effectively maps a protein-folding event (S-peptide/S-protein re-association) to a macroscopic readout (percent restoration of enzymatic activity). RNAse is probably the most thoroughly characterized enzyme of the 20th century, providing an incredible foundation of existing thermodynamic, conformational, and kinetic results against which to compare new experimental data and enable quick, valid translation to new systems. A novel helical peptidomimetic molecule aimed at mimicking the 14 residue S-peptide and regenerating enzymatic activity has been designed. This award will enable in silico evaluation of the stability of a small protein in explicit solvent so that designed hybrid proteins can be compared with their control parents so that enhanced thermal stability can be iteratively evaluated prior to expensive rounds of synthesis and experimental measurement. For this work, we will perform 50-100 ns simulations of the BBA and RNAse A proteins in explicit solvent at multiple temperatures to estimate the temperature for thermal denaturation. Quasi-harmonic approximations will be applied to estimate the conformational space. On a Pentium 4 system, it takes 12 CPU hours to simulate one nanosecond of a solvated BBA system. We plan to use 10,000 CPU hours to simulate the BBA systems which includes both wild type and hybrid proteins, and 20,000 CPU hours to simulate the RNASe A systems.