The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

The outer membranes of Gram negative bacteria are the first points of contact these organisms make with their environment. Understanding how composition determines the mechanical properties of this essential barrier is of paramount importance. Therefore, we developed a new computational method to measure the elasticity of transmembrane proteins found in the outer membrane. Using all-atom molecular dynamics simulations of these proteins, we apply a set of external forces to mechanically stress the transmembrane β-barrels. Our results from four representative β-barrels show that outer membrane proteins display elastic properties that are approximately 70 to 190 times stiffer than neat lipid membranes. These findings suggest that outer membrane β-barrels are a significant source of mechanical stability in bacteria. Our all-atom approach further reveals that resistance to radial stress is encoded by a general mechanism that includes stretching of backbone hydrogen bonds and tilting of β-strands with respect to the bilayer normal. This computational framework facilitates an increased theoretical understanding of how varying lipid and protein amounts affect the mechanical properties of the bacterial outer membrane.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the a-helical sensory rhodopsin II and the b-barrel outer membrane protein Wh ave been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques.Despite their differing topologies,b oth proteins have as imilar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic.T he methyl-bearing side chains of both proteins are,o na verage,m ore dynamic in the ps-ns timescale than any soluble protein characterized to date. Accordingly,b oth proteins retain an extraordinary residual conformational entropyi nt he folded state,w hichp rovides ac ounterbalance to the absence of the hydrophobic effect. Furthermore,t he high conformational entropyc ould greatly influence the thermodynamics underlying membrane-protein functions,i ncluding ligand binding,a llostery,and signaling.

A hallmark feature of biological lipid bilayer structure is a depth-dependent polarity gradient largely resulting from the change in water concentration over the angstrom length scale. This gradient is particularly steep as it crosses the membrane interfacial regions where the water concentration drops at least a million-fold along the direction of the bilayer normal. Although local water content is often assumed to be a major determinant of membrane protein stability, the effect of the water-induced polarity gradient upon backbone hydrogen bond strength has not been systematically investigated. We addressed this question by measuring the free energy change for a number of backbone hydrogen bonds in the transmembrane protein OmpW. These values were obtained at 33 backbone amides from hydrogen/deuterium fractionation factors by nuclear magnetic resonance spectroscopy. We surprisingly found that OmpW backbone hydrogen bond energies do not vary over a wide range of water concentrations that are characteristic of the solvation environment in the bilayer interfacial region. We validated the interpretation of our results by determining the hydrodynamic and solvation properties of our OmpW-micelle complex using analytical ultracentrifugation and molecular dynamics simulations. The magnitudes of the backbone hydrogen bond free energy changes in our study are comparable to those observed in water-soluble proteins, the H-segment of the leader peptidase helix used in the von Heijne and White biological scale experiments, and several interfacial peptides. Our results agree with those reported for the transmembrane α-helical portion of the amyloid precursor protein after the latter values were adjusted for kinetic isotope effects. Overall, our work suggests that backbone hydrogen bonds provide modest thermodynamic stability to membrane protein structures and that many amides are unaffected by dehydration within the bilayer.

Membrane reshaping is an essential biological process. The chemical composition of lipid membranes determines their mechanical properties, and thus the energetics of their shape. Hundreds of distinct lipid species make up native bilayers, and this diversity complicates efforts to uncover what compositional factors drive membrane stability in cells. Simplifying assumptions, therefore, are used to generate quantitative predictions of bilayer dynamics based on lipid composition. One assumption commonly used is that "per lipid" mechanical properties are both additive and constant---that they are an intrinsic property of lipids independent of the surrounding composition. Related to this, is the assumption that lipid bulkiness, or "shape" determines its curvature preference, independently of context. In this study, all-atom molecular dynamics simulations on three separate multi-lipid systems were used to explicitly test these assumptions, applying methodology recently developed to isolate properties of single lipids or nanometer-scale patches of lipids. The curvature preference of populations of lipid conformations were inferred from their redistribution on a dynamically fluctuating bilayer. Representative populations were extracted by both structural similarity and semi-automated hidden Markov model analysis. The curvature preferences of lipid dimers were then determined and compared to an additive model that combines the monomer curvature prefer- ence of both the individual lipids. In all three systems, we identified conformational subpopulations of lipid dimers that showed non-additive curvature preference, in each case mediated by a special chemical interaction (e.g., hydrogen bonding). Our study highlights the importance of specific chemical interactions between lipids in multicomponent bilayers and the impact of interactions on bilayer stiffness. We identify two mechanisms of bilayer softening: Diffusional softening, which is driven by the dynamic coupling between lipid distributions and membrane undulations, and conformational softening, which is driven by the inter-conversion between distinct dimeric conformations.