Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins

Abstract

Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…Thus, much of the helix dynamics responsible for the extensive HDX of the SDS state must occur on the cytoplasmic side. It is unfortunate that the spatial resolution of currently existing BR HDX data [68] is insufficient for directly confirming our view that the cytoplasmic side of SDSdenatured BR is more dynamic than the extracellular side. Nonetheless, additional support for this scenario comes from fluorescence spectroscopic data which show that all of the Trp residues close to the extracellular side maintain a nonpolar environment in SDS [56].…”

“…HDX experiments are considerably more challenging in this regard because back-exchange requires the digestion and LC separation steps to be completed in as little as 15 min [6]. The situation is particularly challenging for membrane proteins where digestion efficiencies under HDX conditions tend to be low [68], and where detergents often interfere with the analysis. It is therefore not surprising that membrane protein covalent labeling has become a fairly routine approach, whereas only a handful of HDX studies in this area have appeared over the past few years [28].…”

“…A detailed analysis of these isotope labeling data is beyond the scope of the current work and has been reported elsewhere [60]. Unfortunately, applying the traditional proteolytic digestion/HDX-MS approach to BR is difficult [68], and hence we were unsuccessful in analyzing the BR deuteration behavior in a spatially resolved manner. The pertinent question explored here is in how far the structure and dynamics of the V179M and L93M variants are different from those of wt BR.…”

Structural differences between thermophilic and mesophilic membrane proteins

“…[8][9][10][11][12] Although most hydrogen-bonded sidechain interactions that have been experimentally measured in membrane proteins appear to make relatively modest contributions, [21][22][23][24][25][26][27] some have been measured as high as 1.8 kcal/mol and in theory it is possible that they could be made even stronger. 26,28 Thus, it seems reasonable to expect that the number of transmembrane interhelical hydrogen bonds might increase in thermophiles.…”

Assessing the effect of D59P mutation in the DE loop region in amyloid aggregation propensity of β2‐microglobulin: A molecular dynamics simulation study

“…A loss or gain of even a weak hydrogen bond could alter the biological function of a protein. 45 The secondary structure of a The figure is generated using PyMol protein is highly dependent on the intramolecular hydrogen bonds and a small decrease in the number of hydrogen bonds could affect the secondary structure of a protein and hence its stability. The intramolecular hydrogen bond analysis was performed for wt β2m and D59P.…”