Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate.

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying beta-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of beta-glucosidase at 90 degrees C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90 degrees C with an overall DeltaG degrees of approximately 20 kcal mol(-1). The high temperatures needed to chemically denature P. furiosus beta-glucosidase and the large DeltaG degrees of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of beta-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.

Identifying interactions between transmembrane helices from the adenosine A2A receptor.

The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and Förster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.

Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor.

The human adenosine A2A receptor (A(2A)R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [theta]222/[theta]208 obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A(2A)R at position M193, which is located in the fifth TM domain, noticeably alters A(2A)R monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.

The role of protein and surfactant interactions in membrane-protein crystallization.

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients were measured by self-interaction chromatography in the presence of sodium malonate, sodium formate and ammonium sulfate. Attractive protein-detergent complex (PDC) interactions were observed as the surfactant cloud-point temperature was approached for each salt, suggesting that surfactant interactions may play an important role in promoting PDC crystallization. Dynamic light scattering and tensiometry measurements show that the interaction trends are strongly influenced by micelle structure and surfactant phase behavior, both of which are sensitive to salt and surfactant concentration. Overall, detailed investigations using a combination of experimental techniques can provide insight into the complex nature of PDC interactions, which is essential to developing rational approaches to membrane-protein crystallization.

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor.

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.

Characterization of peptides corresponding to the seven transmembrane domains of human adenosine A2a receptor.

Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.