Stability, Activity and Structure of Adenylate Kinase Mutants

Abstract

Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/ PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures T,,, and melting enthalpy changes AH,. The heat capacity change AC, of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with T, and AH,. Using the wild-type AC, value, Gibbs free energy changes AG at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal a-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.Keywords. Melting temperature ; melting enthalpy ; site-directed mutagenesis ; helix capping ; non-polar core.The relationships between sequence, structure and function in proteins are most important for understanding the 'second genetic code' and evolution at the molecular level, both of which are current research interests. In this context, we explored two types of sequence/structure relationships : helix capping and core perturbation. In the helix capping experiments we tried to achieve stabilization by introducing negative charges at N-terminals and positive charges at C-terminals of a-helices. In order to analyze core plasticity, we added one carbon atom to a highly conserved and three carbon atoms to a less well conserved nonpolar core. For a thorough assessment of the changes the resulting mutants were characterized by several methods.For these analyses we choose a small enzyme, the adenylate kinase (EC 2.7.4.3) from baker's yeast (Saccharomyces cerevisiae) which catalyzes the transfer of the y-phosphate of ATP to AMP in the presence of bivalent cations according to: Mg" . ATP + AMP * Mg" . ADP + ADP. This reaction is essential for cell survival (Noda, 1973). Adenylate kinases have been purified from various sources and analyzed, several spatial struc-

Higher binding affinity of furin for SARS-CoV-2 spike (S) protein D614G mutant could be associated with higher SARS-CoV-2 infectivity

“…However, the substitutions in the study were in the S2 subunit of the S protein and not near the furin cleavage site. The standard folding enthalpy measured at the melting temperature was higher for G614 S protein at 62.6 kcal/mol compared with 60.9 kcal/mol for D614 S protein, indicating that G614 S protein is less stable at 25 C (Spuergin et al, 1995). Substitutions can have either a stabilizing or a destabilizing effect on WT protein folding, whereby folding free energy measurements on RNA viral proteins with high substitution rates have demonstrated less stable protein structures (Wylie and Shakhnovich, 2011).…”

BPPred: A Web‐based computational tool for predicting biophysical parameters of proteins

“…Therefore, all the relationships that Myers et al (1995) observed for DSASA estimated using the glycine-tripeptide model (Shrake and Rupley 1973;Rose et al 1985;Miller et al 1987;Lesser and Rose 1990) are also valid if the change in solvent-accessible surface area is calculated from the present molecular dynamics simulations. These equations can be used to quantify, for example, DSASA from Clarke and Fersht (1993); z Griko and Privalov (1994); aa Filimonov et al (1993); bb Munoz et al (1994); cc Ropson et al (1990); dd Covalt et al (2001); ee Makhatadze et al (1994); ff Dabora and Marqusee (1994); gg Ionescu et al (2000); hh Zhang et al (1993); ii Hu et al (1992); jj Spuergin et al (1995).…”

Increased Binding Affinity of Furin to D614G Mutant S-glycoprotein May Augment Infectivity of the Predominating SARS-CoV-2 Variant

“…Regarding thermodynamic analysis of D614G mutation, S-glycoprotein stability was assessed and it was found that mutant G614 loop, compared with wild type (WT) D614 loop, was a slightly more dynamic structure, marked by relatively less thermal stability and higher vibrational entropy. In agreement with this notion, G614 was found to be less stable than D614 at 25°C, as inferred from standard folding enthalpy [10]. Next, the authors analyzed the interatomic interactions in the G614 mutant S-glycoprotein and showed that the short 2.7-Å H-bond that existed between S1 (Chain A) and S2 (Chain B) side chains in the WT D614 was lost in the mutant G614 S-glycoprotein.…”