Studies that compare proteins from thermophilic and mesophilic organisms can provide insights into ability of thermophiles to function at their high habitat temperatures and may provide clues that enable us to better define the forces that stabilize all proteins. Most of the comparative studies have focused on thermal stability and show, as expected, that thermophilic proteins have higher T m values than their mesophilic counterparts. Although these comparisons are useful, more detailed thermodynamic analyses are required to reach a more complete understanding of the mechanisms thermophilic protein employ to remain folded over a wider range of temperatures. This complete thermodynamic description allows one to generate a stability curve for a protein that defines how the conformational stability (DG) varies with temperature. Here we compare stability curves for many pairs of homologous proteins from thermophilic and mesophilc organisms. Of the basic methods that can be employed to achieve enhanced thermostability, we find that most thermophilic proteins use the simple method that raises the DG at all temperatures as the principal way to increase their T m . We discuss and compare this thermodynamic method with the possible alternatives. In addition we propose ways that structural alterations and changes to the amino acid sequences might give rise to varied methods used to obtain thermostability.Keywords: protein folding; thermodynamics; protein stability curves; thermophiles; thermostability Life exists almost everywhere on the earth, from deep-sea hydrothermal vents to the heights of the Himalayas, from boiling waters of hot springs to the cold expanses of Antarctica. The organisms that inhabit and have adapted to these extreme and diverse environments are often classified by their altered habitat, such as temperature adaptations (psychrophiles to hyperthermophiles), high salinity adaptations (halophiles), pH adaptations (acidophiles and alkaliphiles), and pressure adaptation (barophiles), to name a few groups. In general, these organisms are often called extremophiles and have been of interest to many protein chemists over the years, dating back to early studies by Perutz and colleagues (Perutz and Raidt 1975;Perutz 1978). In case of adaptations to extremes of pH, salinity, and pressure, membrane components and protective small molecules often play an important role (Jaenicke 1991) and these have been studied quite extensively (Yancey et al. 1982;van de Vossenberg et al. 1998). For temperature adaptations, Reprint requests to: J. Martin Scholtz, Department of Molecular and Cellular Medicine, Texas A&M University, College Station, TX 77843-1114, USA; e-mail: jm-scholtz@tamu.edu; fax: (979) 847-9481.Abbreviations: T m , melting temperature or temperature at midpoint of transition from native to denatured state in a thermal denaturation; DG T , free energy of stabilization at a temperature T; DC p , change in heat capacity associated with protein unfolding; DH, change in enthalpy; DS, change in entropy; T...

SUMMARY Using cryo-electron microscopy, we have solved the structure of a novel icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) co-assembled from purified cargo adaptor Sec23–24 and Sec13–31 lattice forming complexes. The coat structure shows a tetrameric assembly of the Sec23–24 adaptor layer that is positioned beneath the vertices and edges of the Sec13–31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 β-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo including procollagen and chylomicrons that are sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a new mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.

We propose a general model for the role of the Hsp90 ATPase cycle in proteostasis in which Aha1 regulates the dwell time of Hsp90 with client by integrating chaperone function and client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions.

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 -1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability.