Assessing the effects of time and spatial averaging in 15N chemical shift/15N-1H dipolar correlation solid state NMR experiments

The effect of time and spatial averaging on ¹⁵N chemical shift/¹H-¹⁵N dipolar correlation spectra, i.e., PISEMA spectra, of -helical membrane peptides and proteins is investigated. Three types of motion are considered: (a) Librational motion of the peptide planes in the -helix; (b) rotation of the helix about its long axis; and (c) wobble of the helix about a nominal tilt angle. A 2ns molecular dynamics simulation of helix D of bacteriorhodopsin is used to determine the effect of librational motion on the spectral parameters. For the time averaging, the rotation and wobble of this same helix are modelled by assuming either Gaussian motion about the respective angles or a uniform distribution of a given width. For the spatial averaging, regions of possible ¹⁵N chemical shift/¹H-¹⁵N dipolar splittings are computed for a distribution of rotations and/or tilt angles of the helix. The computed spectra show that under certain motional modes the ¹⁵N chemical shift/¹H-¹⁵N dipolar pairs for each of the residues do not form patterns which mimic helical wheel patterns. As a result, the unambiguous identification of helix tilt and helix rotation without any resonance assignments or on the basis of a single assignment may be difficult.     

Molecular dynamics simulations of model trans-membrane peptides in lipid bilayers: a systematic investigation of hydrophobic mismatch

Hydrophobic mismatch, which is the difference between the hydrophobic length of trans-membrane segments of a protein and the hydrophobic width of the surrounding lipid bilayer, is known to play a role in membrane protein function. We have performed molecular dynamics simulations of trans-membrane KALP peptides (sequence: GKK(LA)nLKKA) in phospholipid bilayers to investigate hydrophobic mismatch alleviation mechanisms. By varying systematically the length of the peptide (KALP15, KALP19, KALP23, KALP27, and KALP31) and the lipid hydrophobic length (DLPC, DMPC, and DPPC), a wide range of mismatch conditions were studied. Simulations of durations of 50-200 ns show that under positive mismatch, the system alleviates the mismatch predominantly by tilting the peptide and to a smaller extent by increased lipid ordering in the immediate vicinity of the peptide. Under negative mismatch, alleviation takes place by a combination of local bilayer bending and the snorkeling of the lysine residues of the peptide. Simulations performed at a higher peptide/lipid molar ratio (1:25) reveal slower dynamics of both the peptide and lipid relative to those at a lower peptide/lipid ratio (1:128). The lysine residues have favorable interactions with specific oxygen atoms of the phospholipid headgroups, indicating the preferred localization of these residues at the lipid/water interface.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.     

Measurement of thermodynamic parameters for hydrophobic mismatch 2: intermembrane transfer of a transmembrane helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Formation of a DNA mismatch repair complex mediated by ATP

The mismatch repair proteins, MutS and MutL, interact in a DNA mismatch and ATP-dependent manner to activate downstream events in repair. Here, we assess the role of ATP binding and hydrolysis in mismatch recognition by MutS and the formation of a ternary complex involving MutS and MutL bound to a mismatched DNA. We show that ATP reduces the affinity of MutS for mismatched DNA and that the modulation of DNA binding affinity by nucleotide is even more pronounced for MutS E694A, a protein that binds ATP but is defective for ATP hydrolysis. Despite the ATP hydrolysis defect, E694A, like WT MutS, undergoes rapid, ATP-dependent dissociation from a DNA mismatch. Furthermore, MutS E694A retains the ability to interact with MutL on mismatched DNA. The recruitment of MutL to a mismatched DNA by MutS is also observed for two mutant MutL proteins, E29A, defective for ATP hydrolysis, and R266A, defective for DNA binding. These results suggest that ATP binding in the absence of hydrolysis is sufficient to trigger formation of a MutS sliding clamp. However, recruitment of MutL results in the formation of a dynamic ternary complex that we propose is the intermediate that signals subsequent repair steps requiring ATP hydrolysis.     

Influence of hydrophobic mismatch on the catalytic activity of Escherichia coli GlpG rhomboid protease

Rhomboids comprise a broad family of intramembrane serine proteases that are found in a wide range of organisms and participate in a diverse array of biological processes. High-resolution structures of the catalytic transmembrane domain of the Escherichia coli GlpG rhomboid have provided numerous insights that help explain how hydrolytic cleavage can be achieved below the membrane surface. Key to this are observations that GlpG hydrophobic domain dimensions may not be sufficient to completely span the native lipid bilayer. This formed the basis for a model where hydrophobic mismatch Induces thinning of the local membrane environment to promote access to transmembrane substrates. However, hydrophobic mismatch also has the potential to alter the functional properties of the rhomboid, a possibility we explore in the current work. For this purpose, we purified the catalytic transmembrane domain of GlpG into phosphocholine or maltoside detergent micelles of varying alkyl chain lengths, and assessed proteolytic function with a model water-soluble substrate. Catalytic turnover numbers were found to depend on detergent alkyl chain length, with saturated chains containing 10–12 carbon atoms supporting maximal activity. Similar results were obtained in phospholipid bicelles, with no proteolytic activity being detected in longer-chain lipids. Although differences in thermal stability and GlpG oligomerization could not explain these activity differences, circular dichroism spectra suggest that mismatch gives rise to a small change in structure. Overall, these results demonstrate that hydrophobic mismatch can exert an inhibitory effect on rhomboid activity, with the potential for changes in local membrane environment to regulate activity in vivo.     

Altered dynamics of DNA bases adjacent to a mismatch: a cue for mismatch recognition by MutS

The structural deviations as well as the alteration in the dynamics of DNA at mismatch sites are considered to have a crucial role in mismatch recognition followed by its repair utilizing mismatch repair family proteins. To compare the dynamics at a mismatch and a non-mismatch site, we incorporated 2-aminopurine, a fluorescent analogue of adenine next to a G.T mismatch, a C.C mismatch, or an unpaired T, and at several other non-mismatch positions. Rotational diffusion of 2-aminopurine at these locations, monitored by time-resolved fluorescence anisotropy, showed distinct differences in the dynamics. This alteration in the motional dynamics is largely confined to the normally matched base-pairs that are immediately adjacent to a mismatch/ unpaired base and could be used by MutS as a cue for mismatch-specific recognition. Interestingly, the enhanced dynamics associated with base-pairs adjacent to a mismatch are significantly restricted upon MutS binding, perhaps “resetting” the cues for downstream events that follow MutS binding. Recognition of such details of motional dynamics of DNA for the first time in the current study enabled us to propose a model that integrates the details of mismatch recognition by MutS as revealed by the high-resolution crystal structure with that of observed base dynamics, and unveils a minimal composite read-out involving the base mismatch and its adjacent normal base-pairs.     

Induction of changes in the secondary structure of globular proteins by a hydrophobic surface

Circular dichroism, ellipsometry and radiolabeling techniques were employed to study the induction of changes in the secondary structure of BSA, myoglobin and cytochrome C by a hydrophobic surface. The results showed that adsorbed protein molecules lose their ordered native structure in the initial stage of adsorption and the structure appears to be a random or disordered conformation. Protein molecules adsorbed in later stages adopt a more ordered secondary structure ( helix and structure). The changes of secondary structure of globular proteins induced by a hydrophobic surface can be explained by the steric interaction between adsorbed proteins as well as by hydrophobic interactions during the adsorption process. In addition, there is obviously an intermediate stage in which the protein molecules are mainly in the structure, indicating that for certain proteins, the structure may be a more stable secondary structure than helix on the hydrophobic surface. Correspondence to: S.-F. Sui     

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Na_v1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Na_v1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Na_v1.4. Here, we determine the NMR structure of a selectively ¹³C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By ¹³C NMR, we obtain NMR structural constraints as ¹³C chemical shifts and the ¹H-²H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the ¹H-²H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel Na_vRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Na_v1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.     

Molecular-size corrections to the strength of the hydrophobic effect: a critical review

Large increases in the strength of the hydrophobic effect and, consequently, in the estimates of the hydrophobic contribution to the thermodynamics of protein folding (and other biologically-relevant processes), have been recently advocated on the basis of the application, to model transfer thermodynamic data, of corrections for the solute/solvent size disparity. In this work we first examine the effect of molecular-size corrections on the values calculated from several types of model transfer data. For the transfer of a solute from an organic solvent to water, the above increase is exclusively associated with the application of a solute/water molecular-size correction. Secondly, we critically review and assess the several theoretical arguments that lead to these corrections. In particular, we show that, contrary to previous claims in the literature, the analysis of dissolution processes in terms of ideal-gas, intermediate states does not lead to the molecular-size correction term, but only to expressions equivalent (although not strictly identical) to those derived from the well-known Ben-Naim's statistical-mechanical approach. In general, the several analyses offered or discussed in this work disfavor the application of the solute/water molecular-size corrections.     

Determination of molecular associations of some hydrophobic and hydrophilic bile acids by infrared and Raman spectroscopy

The aim of this study was to analyse the Raman and infrared spectra of eight common mammalian bile acids in order to identify intermolecular interactions between hydroxyl and carbonyl groups. The results are considered in the light of the new hydrophilic/hydrophobic classification of bile acids. The alcohol OH group of the hydrophobic bile acids forms different intermolecular bonds. The most hydrophobic bile acid, lithocholic acid forms polymers, and this may explain its very low water solubility. The hydrophilic bile acids have some of their alcohol OH groups free of any intermolecular interaction. The strongly hydrophilic murideoxycholic acid also forms dimers, again consistent with a very low water solubility. The proposed structural arrangements are in agreement with published crystallographic studies. Received: 7 November 1996 / Accepted: 8 December 1996     

Chlorophyll a is the crucial redox sensor and transmembrane signal transmitter in the cytochrome b6f complex. Components and mechanisms of state transitions from the hydrophobic mismatch viewpoint

The cytochrome (cyt) b₆f complex is involved in the transmembrane redox signaling that triggers state transitions in cyanobacteria and chloroplasts. However, the components and molecular mechanisms are still unclear. In an attempt to solve this long-standing problem, we first focused on the unknown role of a single chlorophyll a (Chla) in cyt b₆f with a new approach based on Chla structural properties. Various b₆f X-ray crystal structures were analyzed to identify their differences, which correlate with differences in Chla molecular volume. We found that the distance of the Rieske [2Fe-2S] cluster to Chla correlates with the distance between a pair of residues at the Q_o-site and the distance between a pair of residues at the opposite membrane side. These correlations were accompanied by the rotation of a key peripheral residue and by changes in the hydrophobic thickness of cyt b₆f. Parallel analysis of cyt bc₁ crystal structures allowed us to conclude that Chla acts as the crucial redox sensor and transmembrane signal transmitter in b₆f for changes in the plastoquinone pool redox state. The hydrophobic mismatch induced by the changed hydrophobic thickness of cyt b₆f is the driving force for the structural reorganizations of the photosynthetic apparatus during induction and the progression of state transitions in cyanobacteria and chloroplasts. A mechanism for LHCII kinase activation in chloroplasts is also proposed. Our understanding of the dynamic structural changes in bc-complexes during turnover at the Q_o-site and state transitions is augmented by the time-sequence ordering of 56 bc crystal structures.     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Charged or Aromatic Anchor Residue Dependence of Transmembrane Peptide Tilt

The membrane-spanning segments of integral membrane proteins often are flanked by aromatic or charged amino acid residues, which may “anchor” the transmembrane orientation. Single spanning transmembrane peptides such as those of the WALP family, acetyl-GWW(LA)_nLWWA-amide, furthermore adopt a moderate average tilt within lipid bilayer membranes. To understand the anchor residue dependence of the tilt, we introduce Leu-Ala “spacers” between paired anchors and in some cases replace the outer tryptophans. The resulting peptides, acetyl-GX²ALW(LA)₆LWLAX²²A-amide, have Trp, Lys, Arg, or Gly in the two X positions. The apparent average orientations of the core helical sequences were determined in oriented phosphatidylcholine bilayer membranes of varying thickness using solid-state ²H NMR spectroscopy. When X is Lys, Arg, or Gly, the direction of the tilt is essentially constant in different lipids and presumably is dictated by the tryptophans (Trp⁵ and Trp¹⁹) that flank the inner helical core. The Leu-Ala spacers are no longer helical. The magnitude of the apparent helix tilt furthermore scales nicely with the bilayer thickness except when X is Trp. When X is Trp, the direction of tilt is less well defined in each phosphatidylcholine bilayer and varies up to 70° among 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine bilayer membranes. Indeed, the X = Trp case parallels earlier observations in which WALP family peptides having multiple Trp anchors show little dependence of the apparent tilt magnitude on bilayer thickness. The results shed new light on the interactions of arginine, lysine, tryptophan, and even glycine at lipid bilayer membrane interfaces.     

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and—together with laterally-aligned tilted amphipathic helices—generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.     

Engineering the hydrophobic residues of a GH11 xylanase impacts its adsorption onto lignin and its thermostability

This study aimed to characterise the parameters governing the non-specific adsorption of a xylanase from Thermobacillus xylanilyticus (Tx-Xyn11) onto lignin isolated from maize stems. Such adsorption may be due to hydrophobic interactions between Tx-Xyn11 and lignin. Our strategy was to mutate hydrophobic residues present on the surface of Tx- Xyn11 into non-hydrophobic residues. Three mutants (P1, P2, and P3) with altered hydrophobic regions were produced and characterised. The thermostability of the P1 mutant was largely decreased compared with the thermostable Tx-Xyn11. The rate of adsorbed enzyme onto lignin was reduced to a similar extent for the P1 and P2 mutants, whereas the adsorption of the P3 mutant was less affected compared with that of Tx-Xyn11. When considered separately, the hydrophobic residues did not affect xylanase adsorption onto lignin. The addition of Tween 20 also led to the decreased adsorption of Tx-Xyn11 onto lignin. These results suggest that hydrophobic interactions are a key parameter in the interaction of Tx-Xyn11 with isolated lignin.