Sequence-Specific Conformational Flexibility of SNARE Transmembrane Helices Probed by Hydrogen/Deuterium Exchange

Abstract

SNARE proteins mediate fusion of intracellular eukaryotic membranes and their alpha-helical transmembrane domains are known to contribute to lipid bilayer mixing. Synthetic transmembrane domain peptides were previously shown to mimic the function of SNARE proteins in that they trigger liposome fusion in a sequence-specific fashion. Here, we performed a detailed investigation of the conformational dynamics of the transmembrane helices of the presynaptic SNAREs synaptobrevin II and syntaxin 1a. To this end, we recorded deuterium/hydrogen-exchange kinetics in isotropic solution as well as in the membrane-embedded state. In solution, the exchange kinetics of each peptide can be described by three different classes of amide deuteriums that exchange with different rate constants. These are likely to originate from exchange at different domains of the helices. Interestingly, the rate constants of each class vary with the TMD sequence. Thus, the exchange rate is position-specific and sequence-specific. Further, the rate constants correlate with the previously determined membrane fusogenicities. In membranes, exchange is retarded and a significant proportion of amide hydrogens are protected from exchange. We conclude that the conformational dynamics of SNARE TMD helices is mechanistically linked to their ability to drive lipid mixing.

Beyond Anchoring: the Expanding Role of the Hendra Virus Fusion Protein Transmembrane Domain in Protein Folding, Stability, and Function

“…Nonpolar ␤-branched residues (valine and isoleucine) are traditionally thought of as being disruptive of ␣-helical structures; however, studies of synthetic peptides in detergent micelles demonstrated that these residues are incorporated into hydrophobic ␣-helices as easily as leucine (39,40). Additionally, as ␤-branched residues are thought to add conformational plasticity to membrane ␣-helices (33,54,72), these residues could be important for driving lipid disordering and later steps of membrane fusion. Thus, inclusion of these residues within viral TMDs may both facilitate promotion of ␣-helical structure and impart a degree of structural flexibility which alanine or residues of similar ␣-helical propensity could not.…”

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

“…Mass spectrometry (MS) is increasingly being used for various types of IMP studies [1,[17][18][19][20][21][22][23][24][25][26][27][28]. Covalent labeling coupled with MS [29] can provide structural information on IMPs.…”

Sequence‐dependent backbone dynamics of a viral fusogen transmembrane helix

“…1 TMDs that were designed to contain Val and Leu at different ratios. 3 Higher membrane fusogenicity correlates with enhanced backbones dynamics, that is with the extent of local and transient helix unfolding, 4,5 which is in accord with the known helix-destabilizing role of b-branched residues.…”

“…The advantage of recording DHX kinetics in isotropic solution is that all amide hydrogen atoms are accessible to solvent while a membrane shields part of a transmembrane peptide from exchange. 4,5 Circular dichroism (CD) spectroscopy revealed the characteristic line shapes diagnostic of a-helices with minima at 208 and 222 nm. Quantitative evaluation of the spectra indicates a-helix contents $70% [ Fig.…”