Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane ␤-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.

In this presentation we illustrate in detail the folding mechanism of a prototypical beta-hairpin, namely the C-terminal fragment of protein GB1, by means of all-atom molecular dynamics (MD) simulations in explicit solvent, using metadynamics to accelerate the sampling of standard MD and reconstruct the free energy of the process. Our results show clearly that the unfolded ensemble of this protein does not comply with the classical view of a collection of disordered coil conformations. Indeed we found out that a fully stretched configuration is unstable towards the formation of a turn in the central region of the peptide. This loop can assume two different conformations: a native-like one, which eventually leads to the 2:4 native structure, and a non-native turn which characterizes the ensemble of the unfolded states among which an ordered 3:5 misfolded structure is particularly stable. Our results, corroborated by several experiments, support the growing idea that the unfolded ensemble of proteins and even of small polypeptides can be characterized by some form of non-native structure.

Experimental results, measured on dimpled test surfaces placed on one wall of different rectangular channels, are given for a ratio of air inlet stagnation temperature to surface temperature of approximately 0.94, and Reynolds numbers based on channel height from 9940 to 74,800. The data presented include friction factors, local Nusselt numbers, spatially averaged Nusselt numbers, and globally averaged Nusselt numbers. The ratios of dimple depth to dimple print diameter δ∕D are 0.1, 0.2, and 0.3 to provide information on the influences of dimple depth. The ratio of channel height to dimple print diameter is 1.00. At all Reynolds numbers considered, local spatially resolved and spatially averaged Nusselt number augmentations increase as dimple depth increases (and all other experimental and geometric parameters are held approximately constant). These are attributed to (i) increases in the strengths and intensity of vortices and associated secondary flows ejected from the dimples, as well as (ii) increases in the magnitudes of three-dimensional turbulence production and turbulence transport. The effects of these phenomena are especially apparent in local Nusselt number ratio distributions measured just inside of the dimples and just downstream of the downstream edges of the dimples. Data are also presented to illustrate the effects of Reynolds number and streamwise development for δ∕D=0.1 dimples. Significant local Nusselt number ratio variations are observed at different streamwise locations, whereas variations with the Reynolds number are mostly apparent on flat surfaces just downstream of individual dimples.

SummaryWe have investigated self-association propensities of aqueous unfolded (U AQ ) forms of eight outer membrane proteins, OmpA, OmpW, OmpX, PagP, OmpT, OmpLa, FadL and Omp85. We found that high urea concentrations maintain all of these OMPs as monomers and that OmpA and OmpX remain monomeric upon dilution to 1M urea. A pH screen showed that basic pH supports the least amount of U AQ OMP self-association, consistent with earlier studies showing that basic pH was optimal for better folding efficiencies. The addition of KCl increased U AQ OMP self-association, although the magnitudes of the responses were varied. These studies showed that urea can be used to tune the amount of U AQ OMP self-association and indicate that the presence of some urea may be useful in optimizing folding conditions because it diminishes aggregation.

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.