Simulation, Experiment, and Evolution: Understanding Nucleation in Protein S6 Folding

In this study, we explore nucleation and the transition state ensemble of the ribosomal protein S6 using a Monte Carlo Go model in conjunction with restraints from experiment. The results are analyzed in the context of extensive experimental and evolutionary data. The roles of individual residues in the folding nucleus are identified and the order of events in the S6 folding mechanism is explored in detail. Interpretation of our results agrees with, and extends the utility of, experiments that shift f-values by modulating denaturant concentration and presents strong evidence for the realism of the mechanistic details in our Monte Carlo Go model and the structural interpretation of experimental f-values. We also observe plasticity in the contacts of the hydrophobic core that support the specific nucleus. For S6, which binds to RNA and protein after folding, this plasticity may result from the conformational flexibility required to achieve biological function. These results present a theoretical and conceptual picture that is relevant in understanding the mechanism of nucleation in protein folding.Comment: PNAS in pres

Phi-values in protein folding kinetics have energetic and structural components

Phi-values are experimental measures of how the kinetics of protein folding is changed by single-site mutations. Phi-values measure energetic quantities, but are often interpreted in terms of the structures of the transition state ensemble. Here we describe a simple analytical model of the folding kinetics in terms of the formation of protein substructures. The model shows that Phi-values have both structural and energetic components. In addition, it provides a natural and general interpretation of "nonclassical" Phi-values (i.e., less than zero, or greater than one). The model reproduces the Phi-values for 20 single-residue mutations in the alpha-helix of the protein CI2, including several nonclassical Phi-values, in good agreement with experiments.Comment: 15 pages, 3 figures, 1 tabl

Conformations of Proteins in Equilibrium

We introduce a simple theoretical approach for an equilibrium study of proteins with known native state structures. We test our approach with results on well-studied globular proteins, Chymotrypsin Inhibitor (2ci2), Barnase and the alpha spectrin SH3 domain and present evidence for a hierarchical onset of order on lowering the temperature with significant organization at the local level even at high temperatures. A further application to the folding process of HIV-1 protease shows that the model can be reliably used to identify key folding sites that are responsible for the development of drug resistance .Comment: 6 pages, 3 eps figure

Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH

In the present study, we have investigated the mechaases, a class that includes the eukaryotic 26S proteanism of degradation by FtsH. We previously showed that some and its structurally related prokaryotic counterFtsH recognized and degraded proteins with nonpolar parts. In the bacterium E. coli, five such ATP-dependent carboxy-terminal tails in vivo (Herman et al., 1998). Two proteases have thus far been identified: ClpAP, ClpXP, classes of nonpolar tails were identified: those recogHslUV, Lon, and FtsH (HflB). Among these, FtsH is the nized solely by FtsH in vivo (e.g., 108; to their overall thermodynamic stability. This was also FtsH is a membrane-anchored metallo-protease with true for the degradation of a membrane protein and its its active site facing the cytoplasm. It contains a wellvariants studied in vivo. Thus, unlike other well-studied conserved 200 amino acid motif called the "AAA" motif ATP-dependent proteases, FtsH appears to lack robust (so named because the diverse functions of its member unfoldase activity. Instead, our experiments are consisproteins are ATPase associated with activity [reviewed tent with the idea that ATP hydrolysis by FtsH is mainly in Ogura and Wilkinson, 2001]). FtsH degrades both inteused to translocate unfolded substrates sequentially gral membrane and cytoplasmic proteins. FtsH performs from the recognition signal to the active site. We propose that lack of a robust unfoldase enables FtsH to discriminate among proteins based on their thermodynamic sta

Protein structures and optimal folding emerging from a geometrical variational principle

Novel numerical techniques, validated by an analysis of barnase and chymotrypsin inhibitor, are used to elucidate the paramount role played by the geometry of the protein backbone in steering the folding to the correct native state. It is found that, irrespective of the sequence, the native state of a protein has exceedingly large number of conformations with a given amount of structural overlap compared to other compact artificial backbones; moreover the conformational entropies of unrelated proteins of the same length are nearly equal at any given stage of folding. These results are suggestive of an extremality principle underlying protein evolution, which, in turn, is shown to be associated with the emergence of secondary structures.Comment: Revtex, 5 pages, 5 postscript figure

Structural determinants of PINK1 topology and dual subcellular distribution

<p>Abstract</p> <p>Background</p> <p>PINK1 is a mitochondria-targeted kinase that constitutively localizes to both the mitochondria and the cytosol. The mechanism of how PINK1 achieves cytosolic localization following mitochondrial processing remains unknown. Understanding PINK1 subcellular localization will give us insights into PINK1 functions and how mutations in PINK1 lead to Parkinson's disease. We asked how the mitochondrial localization signal, the transmembrane domain, and the kinase domain participate in PINK1 localization.</p> <p>Results</p> <p>We confirmed that PINK1 mitochondrial targeting signal is responsible for mitochondrial localization. Once inside the mitochondria, we found that both PINK1 transmembrane and kinase domain are important for membrane tethering and cytosolic-facing topology. We also showed that PINK1 dual subcellular distribution requires both Hsp90 interaction with the kinase domain and the proteolysis at a cleavage site downstream of the transmembrane domain because removal of this cleavage site completely abolished cytosolic PINK1. In addition, the disruption of the Hsp90-PINK1 interaction increased mitochondrial PINK1 level.</p> <p>Conclusion</p> <p>Together, we believe that once PINK1 enters the mitochondria, PINK1 adopts a tethered topology because the transmembrane domain and the kinase domain prevent PINK1 forward movement into the mitochondria. Subsequent proteolysis downstream of the transmembrane domain then releases PINK1 for retrograde movement while PINK1 kinase domain interacts with Hsp90 chaperone. The significance of this dual localization could mean that PINK1 has compartmental-specific functions.</p