title: Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.
abstract: Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: mkuravsky@gmail.com.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kuravsky
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: kmerkushina@gmail.com.
authors.forename: Kseniya
authors.initials: K
authors.lastname: Barinova
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: greyerg@yandex.ru.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Marakhovskaya
authors.affiliation: Bioengineering Center, Russian Academy of Science, prosp. 60-letiya Oktyabrya, 7, korp. 1, Moscow 117312, Russia. Electronic address: meldarov@mail.ru.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Eldarov
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: psemenyuk@belozersky.msu.ru.
authors.forename: Pavel
authors.initials: P
authors.lastname: Semenyuk
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: vimuronets@belozersky.msu.ru.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Muronetz
authors.affiliation: Belozersky Institute of Physico-Chemical Biology of Lomonosov Moscow State University, Leninskie gory, Moscow 119234, Russia. Electronic address: shmal@belozersky.msu.ru.
authors.forename: Elena
authors.initials: E
authors.lastname: Schmalhausen
keywords: Glyceraldehyde-3-phosphate dehydrogenase; Proline rule; Protein stability; Salt bridge