SNAREs can promote complete fusion and hemifusion as alternative outcomes.

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run-like events.

X-ray structure of a water-soluble analog of the membrane protein phospholamban: sequence determinants defining the topology of tetrameric and pentameric coiled coils.

Phospholamban (PLB) is a pentameric transmembrane protein that regulates the Ca(2+)-dependent ATPase SERCA2a in sarcoplasmic reticulum membranes. We previously described the computational design of a water-soluble variant of phospholamban, WSPLB, which reproduced many of the structural and functional properties of the native membrane-soluble protein. While the full-length WSPLB forms a pentamer in solution, a truncated variant forms very stable tetramers. To obtain insight into the tetramer-pentamer cytoplasmic switch, we solved the crystal structure of the truncated construct, WSPLB 21-52. This peptide has a heptad sequence repeat with Leu residues at a- and Ile at d-positions from residues 31-52. The crystal structure revealed that WSPLB 21-52 adopted an antiparallel tetrameric coiled coil. This topology contrasts with the parallel topology of an analogue of the coiled-coil of GCN4 with the same Leu(a) Ile(d) repeat. Analysis of these structures revealed how the nature of the partially exposed residues at e- and g-positions influence the topology formed by the bundle. We also constructed a model for the pentameric form of PLB using the coiled-coil parameters derived from a single monomer in the tetrameric structure. This model suggests that both buried and interfacial hydrogen bonds are important for stabilizing the parallel pentamer.

Computational design of water-soluble analogues of the potassium channel KcsA.

Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in Escherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.

Computational design of a water-soluble analog of phospholamban.

Membrane proteins and water-soluble proteins share a similar core. This similarity suggests that it should be possible to water-solubilize membrane proteins by mutating only their lipid-exposed residues. We have developed computational tools to design water-soluble variants of helical membrane proteins, using the pentameric phospholamban (PLB) as our test case. To water-solublize PLB, the membrane-exposed positions were changed to polar or charged amino acids, while the putative core was left unaltered. We generated water-soluble phospholamban (WSPLB), and compared its properties to its predecessor PLB. In aqueous solution, WSPLB mimics all of the reported properties of PLB including oligomerization state, helical structure, and stabilization upon phosphorylation. We also characterized the truncated mutant WSPLB (21-52) comprising only the former transmembrane segment of PLB. This peptide shows a decreased specificity for forming a pentameric oligomerization state.