No abstract

Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987–2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed “RosettaScripts” for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta’s ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.

Carbon hydrogen bonds between Cα-H donors and carbonyl acceptors are frequently observed between transmembrane helices (Cα-H···O=C). Networks of these interactions occur often at helix−helix interfaces mediated by GxxxG and similar patterns. Cα-H hydrogen bonds have been hypothesized to be important in membrane protein folding and association, but evidence that they are major determinants of helix association is still lacking. Here we present a comprehensive geometric analysis of homodimeric helices that demonstrates the existence of a single region in conformational space with high propensity for Cα-H···O=C hydrogen bond formation. This region corresponds to the most frequent motif for parallel dimers, GAS right , whose best-known example is glycophorin A. The finding suggests a causal link between the high frequency of occurrence of GAS right and its propensity for carbon hydrogen bond formation. Investigation of the sequence dependency of the motif determined that Gly residues are required at specific positions where only Gly can act as a donor with its "side chain" Hα. Gly also reduces the steric barrier for nonGly amino acids at other positions to act as Cα donors, promoting the formation of cooperative hydrogen bonding networks. These findings offer a structural rationale for the occurrence of GxxxG patterns at the GAS right interface. The analysis identified the conformational space and the sequence requirement of Cα-H···O=C mediated motifs; we took advantage of these results to develop a structural prediction method. The resulting program, CATM, predicts ab initio the known high-resolution structures of homodimeric GAS right motifs at near-atomic level.interaction motifs | protein prediction

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.