Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a ß-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Role of protein-phosphorylation events in the anoxia signal-transduction pathway leading to the inhibition of total protein synthesis in isolated hepatocytes.

Incubation of isolated hepatocytes under N2/CO2 (no O2) produced a rapid and strong inhibition of overall polypeptide biosynthesis, which was neither related to cell death nor to the appearance of specific stress proteins. Treatment of the cells with the tyrosine-kinase inhibitor genistein or with the serine/threonine-protein-kinase inhibitor H7 did not modify the impairment of protein synthesis induced by oxygen deprivation, indicating that such signal-transduction pathways are probably not involved in the anoxia-mediated effect. Okadaic acid (100 nM) and Na3VO4 (1 mM) reduced the incorporation of [14C]Leu into proteins of hepatocytes maintained under aerobic conditions (93.3 kPa O2). The effects of oxygen deprivation and okadaic acid were additive, whereas sodium vanadate did not enhance the impairment of protein synthesis induced by anoxia. This observation suggests that a common mechanism, involving the net phosphorylation of protein tyrosine residues, that is insensitive to genistein might participate in the negative control of the translation induced by oxygen deprivation. The effect of anoxia on the synthesis of proteins was fully and rapidly reversible upon the restoration of oxygen supply, thus indicating that hepatocytes are able to sense O2. Although high concentrations of cobalt chloride partially mimic the effect of oxygen deprivation on protein biosynthesis, the nature of such an oxygen sensor remains unknown, and appears unlikely to be a part of a classic haem protein.