Simulating Protein and Nucleic Acid Dynamics on the Microsecond to Millisecond Timescale

Abstract

natural cavity, and variants with substitutions to polar residues to affect the state of hydration of cavities to study its role in pressure unfolding. For 27 variants studied we obtained (a) crystal structures, (b) thermodynamic stabilities using chemical denaturation, and (c) DV of unfolding measured by pressure denaturation monitored with Trp fluorescence. DV of unfolding were also measured using NMR spectroscopy for select variants. The cavities generally did not affect the structure. Although large enough to hold several waters, water molecules were only detected in the cavities when lined with polar groups. The measured DV of variants was always larger than for the wild-type. A near-linear correlation between the DV measured experimentally and the one calculated from structures illustrates the importance of cavities in pressure sensitivity. A correlation between measured DV and thermodynamic stability (DG) suggests that 1 kcal/mol is lost per 12 mL/mol of increased void volume. This study demonstrates irrefutably the significant contributions cavities make towards the pressure sensitivity of proteins and their effects on internal hydration and structural fluctuations of proteins. 241-PosBoard B21 Crystal Structures of Streptococcus Pyogenes Cas2 Protein at Various pH Conditions Ugeene Jeong.College of agricultural and life sciences, Seoul national university, Seoul, Korea, Republic of. Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) proteins provide RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids in archaea and bacteria. Cas2 protein is one of the two core Cas proteins are present in all types of CRISPR-Cas systems and is required for new spacer integration into CRISPR loci. Cas2 homologues from several CRISPR-Cas subtypes were characterized as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the X-ray crystal structures of Streptococcus pyogenes Cas2 protein at three different pHs (5.6, 6.5, and 7.5), and the results of its nuclease activity assay at varying pHs (6.0-9.0). While S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues suggest structural variability and the flexible nature of its putative hinge regions, supporting the supposition that conformational change is important for catalysis. Clustered regularly interspaced short palindromic repeats (CRISPRs) are repetitive genetic elements found in archaeal and bacterial genomes that are involved in RNA-mediated adaptive immunity against foreign invading nucleic acids such as phages and plasmids. CRISPR-associated (cas) genes are also found adjacent to CRISPR loci and encode Cas proteins with a variety of nucleic acid-related functions. Cas5d proteins are subtype I-C s...

Explicit Solvent Hydration Benchmark for Proteins with Application to the PBSA Method

“…Notable attempts to validate implicit solvent models in the context of protein hydration are based on conformational analysis. They either assess the ability of the polypeptide chain to fold into its native conformation in the implicit solvent environment 23,25,58,59 or compare important regions of the free energy landscape derived from explicit and implicit solvent simulations. 60−62 Such studies lead to somewhat mixed conclusions.…”

“…60−62 Such studies lead to somewhat mixed conclusions. On the one hand, a number of small proteins were shown to be able to fold into correct geometries 25 or remain stable in native conformations. 23,58 On the other hand, the results concerning apparently less stable structures, for which the actual conformation may be more dependent on the interplay between internal and solvation energies, are less consistent.…”