pKa Values Of Amino Acids Research Articles

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn(2+) to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn(2+) (ΔZ < 0.44 ± 0.07 per additional Zn(2+) ). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.

Molecular evolution of the sulphite efflux gene SSU1 in Saccharomyces cerevisiae

The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient.

Bayesian model aggregation for ensemble‐based estimates of protein pKavalues

This article investigates an ensemble-based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid pKa predictions. Structure-based pKa calculations play an important role in the mechanistic interpretation of protein structure and are also used to determine a wide range of protein properties. A diverse set of methods currently exist for pKa prediction, ranging from empirical statistical models to ab initio quantum mechanical approaches. However, each of these methods are based on a set of conceptual assumptions that can effect a model's accuracy and generalizability for pKa prediction in complicated biomolecular systems. We use BMA to combine eleven diverse prediction methods that each estimate pKa values of amino acids in staphylococcal nuclease. These methods are based on work conducted for the pKa Cooperative and the pKa measurements are based on experimental work conducted by the García-Moreno lab. Our cross-validation study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods with improvements ranging from 45 to 73% over other method classes. This study also compares BMA's predictive performance to other ensemble-based techniques and demonstrates that BMA can outperform these approaches with improvements ranging from 27 to 60%. This work illustrates a new possible mechanism for improving the accuracy of pKa prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy.

Extracellular pH and Regulation of Integrin-Ligand Interactions

In the tumor and wound microenvironments, the extracellular matrix often exhibits acidic extracellular pH. It is well known that acidic pH can strongly affect processes such as cell adhesion and migration. However, the molecular mechanisms governing these effects have not been established. Here, we consider the hypothesis that acidic extracellular pH directly alters the interactions between cell surface integrin receptors and ECM ligands, which are critical to cell adhesion and migration. We employed Multi-Conformation Continuum Electrostatics to predict amino acid pKa values in the integrin avb3 headpiece, and conducted molecular dynamics simulations at acidic and physiological pH to examine the effect of pH on integrin conformational states. Our results suggest that acidic pH promotes opening of the avb3 headpiece, an important step in activation that can enable more effective ligand-receptor association interactions. This has important implications for downstream cell processes in the cancer and wound environments. We also conducted molecular-level experimental approaches, including flow cytometry and atomic force microscope-enabled force spectroscopy, to further examine the role of pH in regulating integrin-ligand interactions. These molecular-level results are connected to cell-level measurements of adhesion and migration at different pH levels, providing a detailed, multi-scale understanding of how acidic extracellular pH affects cell behavior.

Ionization properties of titratable groups in ribonuclease T1. I. pKa values in the native state determined by two-dimensional heteronuclear NMR spectroscopy.

pKa values of amino acid side chains of ribonuclease T1 have been determined from the pH dependence of 13C and 15N resonances. It was possible to derive pKa values of single protonation or deprotonation sites of carboxylate and imidazole groups. Deviations from pKa values of free amino acids could be interpreted with electrostatic interactions of corresponding side chains with the protein environment. In particular, the interaction between H27 and E82 led to an increase of the H27 pKa and a decrease of the E82 pKa. The pKa of E28 at the C-terminal end of the alpha-helix was increased because of the dipolar character of the alpha-helix. D76 did not titrate in the investigated pH range of about 2-9. From the chemical shift value this buried side chain seems to be protonated. The pKa values of side chains in the active site deviate from a normal behaviour. The lower pKa value of E58 may be interpreted with the close proximity of this side chain with positively charged H40 and R77. A novel two-dimensional 1H(13Cdelta)13Cgamma correlation experiment was developed to observe the pH dependence of the chemical shifts of the Cgamma resonances of histidine residues. From the inspection of the Cgamma chemical shift-pH profiles it was possible to determine the predominant tautomeric form for the histidine residues at higher pH values.

Characterization of Three Components of Human Adenovirus Proteinase Activity in Vitro

Human adenovirus contains a virion-associated proteinase activity essential for the development of infectious virus. Maximal proteinase activity in vitro had been shown to require three viral components: the L3 23-kDa protein, an 11-amino acid cofactor (pVIc), and the viral DNA. Here, we present a quantitative purification procedure for a recombinant L3 23-kDa protein (recombinant endoproteinase (rEP)) expressed in Escherichia coli and the procedure that led to the purification and identification of pVIc as a cofactor. The cofactors stimulate proteinase activity not by decreasing Km, which changes by no more than 2-fold, but by increasing kcat. rEP alone had a small amount of activity, the kcat of which increased 355-fold with pVIc and 6072-fold with adenovirus serotype 2 (Ad2) DNA as well. Curves of Vmax of rEP.pVIc complexes with the substrate (Leu-Arg-Gly-NH)2-rhodamine as a function of pH in the absence and presence of Ad2 DNA indicate that the pKa values of amino acids that affect catalysis are quite different from those that affect catalysis by the cysteine proteinase papain. The pKa values in the absence of Ad2 DNA are 5.2, 6.4, 6.9, 7.5, and 9.4, and those in its presence are 5.2, 6.5, 7.4, and 8.8.