Enhanced internal dynamics of a membrane transport protein during substrate translocation.

Conformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of alpha-helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate-loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.

Effects of ligand binding on the internal dynamics of maltose-binding protein.

Ligand binding to proteins often causes large conformational changes. A typical example is maltose-binding protein (MBP), a member of the family of periplasmic binding proteins of Gram-negative bacteria. Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e. the distance between its two domains decreases. In contrast, binding of the larger, nonphysiological ligand beta-cyclodextrin does not result in closure of the binding cleft. We have investigated the dynamic properties of MBP in its different states using time-resolved tryptophan fluorescence anisotropy. We found that the 'empty' protein exhibits strong internal fluctuations that almost vanish upon ligand binding. The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side-chains relative to the protein backbone, and orientational fluctuations of entire domains. After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side-chains become rather restricted. This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger beta-cyclodextrin. The fluctuations of tryptophan side-chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.

Adhesion forces of lipids in a phospholipid membrane studied by molecular dynamics simulations.

Lipid adhesion forces can be measured using several experimental techniques, but none of these techniques provide insight on the atomic level. Therefore, we performed extensive nonequilibrium molecular dynamics simulations of a phospholipid membrane in the liquid-crystalline phase out of which individual lipid molecules were pulled. In our method, as an idealization of the experimental setups, we have simply attached a harmonic spring to one of the lipid headgroup atoms. Upon retraction of the spring, the force needed to drag the lipid out of the membrane is recorded. By simulating different retraction rates, we were able to investigate the high pull rate part of the dynamical spectrum of lipid adhesion forces. We find that the adhesion force increases along the unbinding path, until the point of rupture is reached. The maximum value of the adhesion force, the rupture force, decreases as the pull rate becomes slower, and eventually enters a friction-dominated regime. The computed bond lengths depend on the rate of rupture, and show some scatter due to the nonequilibrium nature of the experiment. On average, the bond length increases from approximately 1.7 nm to 2.3 nm as the rates go down. Conformational analyses elucidate the detailed mechanism of lipid-membrane bond rupture. We present results of over 15 ns of membrane simulations. Implications for the interpretation and understanding of experimental rupture data are discussed.

Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH.

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved fluorescence anisotropy (TRFA) and fluorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM-DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM-DR association involves a larger hydrophobic surface area with DR/class II-associated invariant chain peptides (CLIP) than with stable DR/peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM-DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM-DR complex at the cell surface.

Pathway of detergent-mediated and peptide ligand-mediated refolding of heterodimeric class II major histocompatibility complex (MHC) molecules.

We investigated the mechanism of refolding and reassembly of recombinant alpha and beta chains of the class II major histocompatibility molecules (MHC-II) HLA-DRB5*0101. Both chains were expressed in the cytosol of Escherichia coli, purified in urea and SDS, and reassembled to functional heterodimers by replacement of SDS by mild detergents, incubation in a redox-shuffling buffer and finally by oxidation and removal of detergent. Refolding was mediated by mild detergents and by peptide ligands. Early stages of structure formation were characterized by circular dichroism, fluorescence, and time-resolved fluorescence anisotropy decay (FAD) spectroscopies. We found that formation of secondary structure was detectable after replacement of SDS by mild detergents. At that stage the alpha and beta chains were still monomeric, the buffer was strongly reducing, and the folding intermediates did not yet interact with peptide ligands. Formation of folding intermediates capable of interacting with peptide ligands was detected after adjusting the redox potential with oxidized glutathione and incubation in mild detergents. We conclude that at that stage a tertiary structure close to the native structure is formed at least locally. The nature and concentration of detergent critically determined the refolding efficiency. We compared detergents with different carbohydrate headgroups, and with aliphatic chains ranging from C6 to C14 in length. For each of the detergents we observed a narrow concentration range for mediating refolding. Surprisingly, detergents with long aliphatic chains had to be used at higher concentrations than short-chain detergents, indicating that increasing the solubility of folding intermediates is not the only function of detergents during a refolding reaction. We discuss structure formation and interactions of detergents with stable folding intermediates. Understanding such interactions will help to develop rational strategies for refolding hydrophobic or oligomeric proteins.

Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature.

Molecular dynamics simulations of 500 ps were performed on a system consisting of a bilayer of 64 molecules of the lipid dipalmitoylphosphatidylcholine and 23 water molecules per lipid at an isotropic pressure of 1 atm and 50 degrees C. Special attention was devoted to reproduce the correct density of the lipid, because this quantity is known experimentally with a precision better than 1%. For this purpose, the Lennard-Jones parameters of the hydrocarbon chains were adjusted by simulating a system consisting of 128 pentadecane molecules and varying the Lennard-Jones parameters until the experimental density and heat of vaporization were obtained. With these parameters the lipid density resulted in perfect agreement with the experimental density. The orientational order parameter of the hydrocarbon chains agreed perfectly well with the experimental values, which, because of its correlation with the area per lipid, makes it possible to give a proper estimate of the area per lipid of 0.61 +/- 0.01 nm2.

Investigation of the proton release channel of bacteriorhodopsin in different intermediates of the photo cycle. A molecular dynamics study.

Molecular dynamics simulations on bacteriorhodopsin were performed starting from a conformation based on electron cryomicroscopy studies [Henderson, R., et al. (1990) J. Mol. Biol. 213, 899-929]. We examined the proton release channel in different intermediates of the bacteriorhodopsin photocycle. In the simulations of the ground state, two stable sets of conformations were observed differing in the distance of the guanidinium group of Arg82 to the Schiff base. The set of conformations in which Arg82 is located closer to the Schiff base has a lower potential energy and agrees better with experimental data than the other set. With both sets, we performed a series of simulations in which the chromophore was isomerized to different states using purposive and nonpurposive methods. The energetic consideration of the different states argues for the location of the guanidinium group of Arg82 close to the Schiff base. The results also show that no C13-C14, C14-C15 dicis conformation of the retinal occurs in the K/L-intermediate of the photocycle instead supporting the occurrence of C13-C14 cis in these intermediates. In a last series of simulations, we modeled the M-intermediate of the bacteriorhodopsin photocycle. Again, comparison to different experimental data indicates that Arg82 points toward the Schiff base. We conclude that the guanidinium group of Arg82 is located close to the Schiff base at a distance of approximately 4.5 A and stays there at least up to the M-intermediate of the photocycle.

The use of a long-lifetime component of tryptophan to detect slow orientational fluctuations of proteins.

The membrane protein porin and a synthetic polypeptide of 21 hydrophobic residues were inserted into detergent micelles or lipid membranes, and the fluorescence of their single tryptophan residue was measured in the time-resolved and polarized mode. In all cases, the tryptophan fluorescence exhibits a long-lifetime component of about 20 ns. This long-lifetime component was exploited to detect slow orientational motions in the range of tens of nanoseconds via the anisotropy decay. For this purpose, the analysis of the anisotropy has to be extended to account for different orientations of the dipoles of the short- and long-lifetime components. This is demonstrated for porin and the polypeptide solubilized in micelles, in which the longest relaxation time reflects the rotational diffusion of the micelle. When the polypeptide is inserted into lipid membranes, it forms a membrane-spanning alpha-helix, and the slowest relaxation process is interpreted as reflecting orientational fluctuations of the helix.

Modeling of halorhodopsin and rhodopsin based on bacteriorhodopsin.

Bacteriorhodopsin (BR), halorhodopsin (HR), and rhodopsin (Rh) all belong to the class of seven-helix membrane proteins. For BR, a structural model at atomic resolution is available; for HR, diffraction data are available only down to a resolution of 6 A in the membrane plane, and for Rh, down to 9 A. BR and HR are closely related proteins with a sequence homology of 34%, while Rh does not share any sequence homology with BR. An atomic model for HR is derived that is based on sequence alignment and the atomic model for BR and is improved by molecular dynamics simulations. The model structure obtained accounts well for the experimentally observed difference between HR and BR in the projection map, where HR exhibits a higher density in the region between helices D and E. The reason for this difference lies partially in the different side chains and partially in slightly different helix tilts. The scattering amplitudes and phases of the model structure are calculated and agree with the experimental data down to a resolution of about 8 A. If the helix positions are adopted from the projection map for HR and used as input in the model, this number improves to 7 A. Analogously, an atomic model for Rh is derived based on the atomic model for BR and subjected to molecular dynamics simulations. Optimal agreement with the experimental projection map for Rh is obtained when the entire model structure is rotated slightly about two axes in the membrane plane. The agreement with the experimental projection map is not as satisfactory as for HR, but the results indicate that even for a nonhomologous, but structurally related, protein such as Rh, an acceptable model structure can be derived from the structure of BR.

Proton transport across transient single-file water pores in a lipid membrane studied by molecular dynamics simulations.

To test the hypothesis that water pores in a lipid membrane mediate the proton transport, molecular dynamic simulations of a phospholipid membrane, in which the formation of a water pore is induced, are reported. The probability density of such a pore in the membrane was obtained from the free energy of formation of the pore, which was computed from the average force needed to constrain the pore in the membrane. It was found that the free energy of a single file of water molecules spanning the bilayer is 108(+/-10) kJ/mol. From unconstrained molecular dynamic simulations it was further deduced that the nature of the pore is very transient, with a mean lifetime of a few picoseconds. The orientations of water molecules within the pore were also studied, and the spontaneous translocation of a turning defect was observed. The combined data allowed a permeability coefficient for proton permeation across the membrane to be computed, assuming that a suitable orientation of the water molecules in the pore allows protons to permeate the membrane relatively fast by means of a wirelike conductance mechanism. The computed value fits the experimental data only if it is assumed that the entry of the proton into the pore is not rate limiting.

Folding and membrane insertion of the trimeric beta-barrel protein OmpF.

We have studied folding and membrane insertion of the porin OmpF and compared it to OmpA. Both are beta-barrel membrane proteins from the outer membrane of Escherichia coli, OmpF forming trimers and OmpA monomers. Each of them can be unfolded in solubilized form in a water/urea mixture. Refolding is initiated by dilution into a dispersion of lipid vesicles or lipid/detergent vesicles, whereupon OmpF and OmpA refold and insert into the membranes. Folding and insertion of the monomers proceed in a similar way for the two proteins, but native OmpF appears more slowly and with a lower yield than native OmpA because of trimerization of OmpF. The dependence of the yield of refolding, membrane insertion, and trimerization on pH, lipid concentration, and the presence of detergent was investigated. Trimerization of OmpF is shown to take place at or in the membrane and a membrane-inserted dimer is detected as an intermediate of this process.

Kinetics of folding and membrane insertion of a beta-barrel membrane protein.

We have studied the kinetics of folding and membrane insertion of the outer membrane protein OmpA of Escherichia coli. In the native structure, its membrane-inserted domain forms a beta-barrel. The protein was unfolded in solubilized form in water/urea, and refolding was induced by dilution of urea and simultaneous addition of lipid vesicles. Three transitions along the folding pathway could be distinguished. Their characteristic times lie below a second, in the range of minutes, and in the range of an hour. The fast process corresponds to the transition from the unfolded state in water/urea to a misfolded state in water, the moderately slow process to a transition from the misfolded state to a partially folded state in the membrane, and the slow process to the transition from the partially folded to the native state. The partially folded state in the membrane is interpreted as the analogue of the molten globule state of soluble proteins.

Lipid vesicle adsorption versus formation of planar bilayers on solid surfaces.

The absorption and spreading behavior of lipid vesicles composed of either palmitoyloleoylphosphatidylcholine (POPC) or Escherichia coli lipid upon contact with a glass surface was examined by fluorescence measurements. Fluorescently labeled lipids were used to determine 1) the amount of lipid adsorbed at the surface, 2) the extent of fusion of the vesicles upon contact with the surface, 3) the ability of the adsorbed lipids to undergo lateral diffusion, and 4) the accessibility of the adsorbed lipids by external water soluble molecules. The results of these measurements indicate that POPC vesicles spread on the surface and form a supported planar bilayer, whereas E. coli lipid vesicles adsorb to the surface and form a supported vesicle layer. Supported planar bilayers were found to be permeable for small molecules, whereas supported vesicles were impermeable and thus represented immobilized, topologically separate compartments.

A nontransportable substrate for lactose permease.

A substrate for lactose permease of Escherichia coli was synthesized that binds to the protein with a relatively high affinity, but is not transported to any detectable extent. This substrate, 6'-[(N-phenylalanylphenylalanyl)amino]hexyl 1-thio-beta-D-galactoside, is a peptide galactoside composed of a bulky aromatic dipeptide that is linked to galactose via an aminohexyl spacer. Binding of the peptide galactoside to lactose permease in cytoplasmic membranes was determined in a competition assay yielding a dissociation constant of 150 microM. Transport was measured by a counterflow assay using lipid vesicles with reconstituted lactose permease. An upper limit for the rate constant of transport was obtained as 0.02 s-1, 3 orders of magnitude smaller than the value for lactose.

Structure and fluctuations of bacteriorhodopsin in the purple membrane: a molecular dynamics study.

Molecular dynamics simulations on bacteriorhodopsin were performed starting from the model structure described by Henderson et al. The simulations were gradually improved by first treating a monomer in vacuum and then adding further monomers, lipids, and water to finally simulate a unit cell of the hexagonal lattice of the purple membrane containing a trimer and lipids and water on both sides. During all simulations, the protein structure moved away from the model structure to reach a root-mean-square (r.m.s.) deviation of 2 to 3 A. In the simulations with the trimer, the structures of the three monomers differed by about the same amount and averaging over them led to an average structure with a considerably smaller r.m.s. deviation. The best average structure obtained had an r.m.s. deviation from the model structure of 1.3 A. Fluctuations of the protein, the lipids, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops. Unexpected, however, was the finding that the fluctuations of the protein are asymmetric with respect to the midplane of the membrane. The fluctuations of the loops and the ends of the helices on the inner side of the membrane are much stronger than on the outer side. This asymmetry is also reflected by the fluctuations for the lipids, the lipids of the inner leaflet fluctuating more strongly than those of the outer leaflet. The asymmetry was observed only in the presence of water on both sides of the membrane. On the average, nine water molecules were found inside the protein, most of them undergoing exchange with external water.

Characterization of two membrane-bound forms of OmpA.

The insertion of the outer membrane protein A (OmpA) into lipid bilayers was studied by limited proteolysis, polarized Fourier transform infrared (FTIR) spectroscopy, and fluorescence spectroscopy. In the native state, OmpA is thought to form a barrel of eight antiparallel beta-strands. For the present study, it was isolated in an unfolded form, purified, and exposed to performed vesicles of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and three phospholipids that were brominated in different positions of their sn-2 chains (4,5-BrPC, 9,10-BrPC, and 11,12-BrPC). Limited proteolysis revealed two membrane-bound forms of OmpA, namely an "adsorbed" (35 kDa) and an "inserted" (30 kDa) form [Surrey, T., & Jähnig, F. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7457-7461]. Which form was found after membrane binding and refolding depended on the lipids used and on the temperature. Polarized attenuated total reflection (ATR)-FTIR spectra were recorded with OmpA bound to germanium-supported bilayers in both forms. The position of the amide I' band indicated quite large fractions of beta-structure of OmpA in both membrane-bound forms (35-45% in the adsorbed form and 45-55% in the inserted form). Measurements of the linear dichroism of the amide I' bands in the inserted form are consistent with an antiparallel beta-barrel in which the strands are inclined at about 36 degrees from the membrane normal. The average angle of the beta-strands to the bilayer normal is likely larger in the 35 kDa form than in the inserted form.(ABSTRACT TRUNCATED AT 250 WORDS)

A long lifetime component in the tryptophan fluorescence of some proteins.

The tryptophan fluorescence of two membrane proteins (outer membrane protein A and lactose permease), a 21-residue hydrophobic peptide, three soluble proteins (rat serum albumin, ribonuclease T1, and azurin), and N-acetyltryptophanamide (NATA) was investigated by time-resolved measurements extended over 65 ns. A long lifetime component with a characteristic time of 25 ns and an amplitude below 1% was found for outer membrane protein A, lactose permease, the peptide in lipid membranes, and azurin in water, but not for rat serum albumin, ribonuclease T1, and NATA in water. When outer membrane protein A was dissolved and unfolded in guanidinum hydrochloride, the long lifetime component disappeared. Hence, a hydrophobic environment seems to be a necessary requirement for the long lifetime component to be present. However, NATA dissolved in butanol does not exhibit the long lifetime component, while the peptide dissolved in the same solvent under conditions which preserve its helical structure does show the long lifetime. Thus, a regular secondary structure for the polypeptide chain to which the tryptophan residue belongs seems to be a second necessary requirement for the long lifetime component to be present. The long lifetime component may therefore be seen in the context of protein substates.