pH-Dependent Conformational Changes Due to Ionizable Residues in a Hydrophobic Protein Interior: The Study of L25K and L125K Variants of SNase

Abstract

Ionizable residues in the hydrophobic interior of certain proteins are known to play important roles in life processes like energy transduction and enzyme catalysis. These internal ionizable residues show experimental apparent pK a values having large shifts as compared to their values in solution.In the present work, we study the pH-dependent conformational changes undergone by two variants of staphylococcal nuclease (SNase), L25K and L125K, using pH replica exchange molecular dynamics (pH-REMD) in explicit solvent. Our results show that the observed pK a of Lys25 and Lys125 are significantly different than their pK a in solution. We observed that the internal lysine residues prefer to be water-exposed when protonated at low pH, but they remain buried within the hydrophobic pocket when deprotonated at high pH. Using thermodynamic laws, we estimate the microscopic conformation-specific pK a of the water-exposed and buried conformations of the internal lysine residues and explain their relation to the macroscopic observed pK a values. We present the differences in the microscopic mechanisms that lead to similar experimentally observed apparent pK a of Lys25 and Lys125, and explain the need of thermodynamic models of different complexities to account for our calculations. We see that L25K displays pH-dependent fluctuations throughout the entire β barrel and the α1 helix. In contrast, pH-independent fluctuations are observed in L125K, primarily limited to the α3 helix. The present computational study offers a detailed atomistic understanding of the determinants of the observed anomalous pK a of internal ionizable residues, bolstering the experimental findings.

Scalable molecular dynamics on CPU and GPU architectures with NAMD

“…This is an increasingly popular approach for directly probing different protonation states in biomolecules. Indeed, several methods and implementations have been reported, 160,[167][168][169][170][171][172] and employed to study pH effects on molecular conformation, 173 ligand binding, 174,175 as well as enzymatic activity. 176 In practice, how these calculations are carried out and subsequently analyzed can depend rather significantly on the specific methodology being used.…”

pH Effects and Cooperativity among Key Titratable Residues for Escherichia coli Glycinamide Ribonucleotide Transformylase

“…In the previous section, we defined the thermodynamic model for the CCPS residues along with the buried conformation of the folate loop. We are building upon the work of Bevilacqua’s model on ribozymes , and Sarkar’s model on SNase to study the pH-dependent behavior for the E. coli GAR Tfase enzyme.…”

“…In the previous section, we defined the thermodynamic model for the CCPS residues along with the buried conformation of the folate loop. We are building upon the work of Bevilacqua's model on ribozymes 40,41 and Sarkar's model on SNase 75 to study the pH-dependent behavior for the E. coli GAR Tfase enzyme. This thermodynamic model will determine the microscopic pK a values of CCPS residues in the catalytically active form of the E. coli GAR Tfase enzyme.…”

pH-Dependent Conformational Changes Lead to a Highly Shifted pKa for a Buried Glutamic Acid Mutant of SNase

“…We also calculated the solvent-accessible surface area (SASA) of the COOH terminal of the Glu23 side chain using a probe radius of 1.4 Å at each pH (Figure S3). Similar to the treatment in the L25K variant of SNase, a cutoff SASA criterion of the Glu23 side chain has been used to separate the conformational ensemble of V23E into buried and water-exposed states. All the snapshots of Glu23 with SASA values less than or equal to 5 Å 2 were considered to be “buried”.…”