Molecular mechanics is powerful for its speed in atomistic simulations, but an accurate force field is required. The Amber ff99SB force field improved protein secondary structure balance and dynamics from earlier force fields like ff99, but weaknesses in side chain rotamer and backbone secondary structure preferences have been identified. Here, we performed a complete refit of all amino acid side chain dihedral parameters, which had been carried over from ff94. The training set of conformations included multidimensional dihedral scans designed to improve transferability of the parameters. Improvement in all amino acids was obtained as compared to ff99SB. Parameters were also generated for alternate protonation states of ionizable side chains. Average errors in relative energies of pairs of conformations were under 1.0 kcal/mol as compared to QM, reduced 35% from ff99SB. We also took the opportunity to make empirical adjustments to the protein backbone dihedral parameters as compared to ff99SB. Multiple small adjustments of φ and ψ parameters were tested against NMR scalar coupling data and secondary structure content for short peptides. The best results were obtained from a physically motivated adjustment to the φ rotational profile that compensates for lack of ff99SB QM training data in the β-ppII transition region. Together, these backbone and side chain modifications (hereafter called ff14SB) not only better reproduced their benchmarks, but improved secondary structure content in small peptides, and reproduction of NMR χ1 scalar coupling measurements for proteins in solution. We also discuss the Amber ff12SB parameter set, a preliminary version of ff14SB that includes most of its improvements.

The millisecond time scale needed for molecular dynamics simulations to approach the quantitative study of protein folding is not yet routine. One approach to extend the simulation time scale is to perform long simulations on specialized and expensive supercomputers such as Anton. Ideally, however, folding simulations would be more economical while retaining reasonable accuracy, and provide feedback on structure, stability and function rapidly enough if partnered directly with experiment. Approaches to this problem typically involve varied compromises between accuracy, precision, and cost; the goal here is to address whether simple implicit solvent models have become sufficiently accurate for their weaknesses to be offset by their ability to rapidly provide much more precise conformational data as compared to explicit solvent. We demonstrate that our recently developed physics-based model performs well on this challenge, enabling accurate all-atom simulated folding for 16 of 17 proteins with a variety of sizes, secondary structure, and topologies. The simulations were carried out using the Amber software on inexpensive GPUs, providing ∼1 μs/day per GPU, and >2.5 ms data presented here. We also show that native conformations are preferred over misfolded structures for 14 of the 17 proteins. For the other 3, misfolded structures are thermodynamically preferred, suggesting opportunities for further improvement.

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...