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.     

The determinants of hydrophobic mismatch response for transmembrane helices

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~ 45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.     

Aggregation of model membrane proteins, modulated by hydrophobic mismatch, membrane curvature, and protein class

Aggregation of transmembrane proteins is important for many biological processes, such as protein sorting and cell signaling, and also for in vitro processes such as two-dimensional crystallization. We have used large-scale simulations to study the lateral organization and dynamics of lipid bilayers containing multiple inserted proteins. Using coarse-grained molecular dynamics simulations, we have studied model membranes comprising ∼7000 lipids and 16 identical copies of model cylindrical proteins of either α-helical or β-barrel types. Through variation of the lipid tail length and hence the degree of hydrophobic mismatch, our simulations display levels of protein aggregation ranging from negligible to extensive. The nature and extent of aggregation are shown to be influenced by membrane curvature and the shape or orientation of the protein. Interestingly, a model β-barrel protein aggregates to form one-dimensional strings within the bilayer plane, whereas a model α-helical bundle forms two-dimensional clusters. Overall, it is clear that the nature and extent of membrane protein aggregation is dependent on several aspects of the proteins and lipids, including hydrophobic mismatch, protein class and shape, and membrane curvature.     

Immunoblotting of hydrophobic integral membrane proteins

For diagnosis and research purposes it is frequently desirable to measure by immunoblotting small amounts of proteins in complex mixtures such as tissue biopsy homogenates. Standard immunoblot procedures that give excellent results for soluble proteins unexpectedly gave low and irreproducible signals with some hydrophobic membrane proteins. We found that this was due to inefficient electrophoretic transfer to nitrocellulose, which could be corrected by modification of the transblot buffer. Hydrophobic integral membrane proteins of peroxisomes as well as other rat and human liver proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. The nitrocellulose-bound proteins were detected both by staining and by immunoblotting with an antiserum against the 22-kDa integral membrane protein of peroxisomes plus 125I-labeled protein A. A modified transblot buffer with 0.7 M glycine and 25 mM Tris (pH 7.7) but no methanol allowed use of a much shorter transfer time and strikingly improved the electrophoretic transfer of membrane proteins such that a peroxisomal integral membrane protein could be easily detected in human liver biopsy homogenates.     

A conformational preference parameter to predict helices in integral membrane proteins

Assignments were made for helical regions in several integral membrane proteins using an algorithm devised to delineate the transmembrane helices in bacteriorhodopsin (Eur. J. Biochem. 182 (1982) 565-575). A new conformational preference parameter for membrane-buried helices was obtained. The use of this parameter to predict helices in membrane proteins is discussed. When applied to the L and M subunits of Rhodopseudomonas sphaeroides, five helices were predicted, which is consistent with the three-dimensional X-ray crystal structure. Data on signal sequences and amino acid exchanges in membrane proteins are also analysed and discussed     

Outer membrane of Salmonella typhimurium: Transmembrane diffusion of some hydrophobic substances

The outer membrane, which is composed of lipopolysaccharide, phospholipids, and proteins, is a layer of the cell wall of Gram-negative bacteria, and apparently acts as a penetration barrier for various substances. It had been shown by other workers that “deep rough” mutants of Salmonella typhimurium, whose lipopolysaccharides lack most of the saccharide chains, were much more sensitive than the wild type strain to certain antibiotics and dyes, but not to others. We found that the former group of agents are usually hydrophobic and the latter group mostly hydrophilic. All hydrophilic antibiotics had molecular weights lower than 650, and one of them was shown to diffuse through the outer membrane at 0 °C. In contrast, some hydrophobic antibiotics had molecular weights in excess of 1200, and the rate of diffusion of one of them was shown to be extremely dependent both on temperature and on the structure of lipopolysaccharide present. These data and results presented elsewhere suggest, but do not necessarily prove, that most hydrophilic antibiotics diffuse through aqueous pores, whereas hydrophobic antibiotics and dyes mainly penetrate by dissolving into the hydrocarbon interior of the outer membrane. In contrast to the outer membrane of deep rough mutants, that of the wild type strain and less defective rough mutants was unusual among biological membranes in that it was practically impermeable to hydrophobic agents. It is proposed that the difference in hydrophobic permeability between the two types of strain is due to radical differences in the organization of the outer membrane, more specifically to the presence or absence of exposed phospholipid bilayer regions.     

Conformational preference functions for predicting helices in membrane proteins

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the α-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in α-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 α-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known α-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements. © 1993 John Wiley & Sons, Inc.     

Prediction of buried helices in multispan alpha helical membrane proteins

Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation. Given that the number of buried helices in a membrane protein is known, our method achieves an accuracy of 78% and a Matthew's correlation coefficient of 0.68. A server for this tool (RANTS) is available online at http://gila.bioengr.uic.edu/lab/.     

Transmembrane domain-dependent partitioning of membrane proteins within the endoplasmic reticulum

The length and hydrophobicity of the transmembrane domain (TMD) play an important role in the sorting of membrane proteins within the secretory pathway; however, the relative contributions of protein-protein and protein-lipid interactions to this phenomenon are currently not understood. To investigate the mechanism of TMD-dependent sorting, we used the following two C tail-anchored fluorescent proteins (FPs), which differ only in TMD length: FP-17, which is anchored to the endoplasmic reticulum (ER) membrane by 17 uncharged residues, and FP-22, which is driven to the plasma membrane by its 22-residue-long TMD. Before export of FP-22, the two constructs, although freely diffusible, were seen to distribute differently between ER tubules and sheets. Analyses in temperature-blocked cells revealed that FP-17 is excluded from ER exit sites, whereas FP-22 is recruited to them, although it remains freely exchangeable with the surrounding reticulum. Thus, physicochemical features of the TMD influence sorting of membrane proteins both within the ER and at the ER-Golgi boundary by simple receptor-independent mechanisms based on partitioning.     

Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii

Overlapping mechanisms that function simultaneously in the intracellular sorting of mammalian membrane proteins often confound delineation of individual sorting pathways. By analyzing sorting in the evolutionarily simpler organism Toxoplasma gondii, we demonstrate a role for transmembrane domain (TMD) length in modulating the signal-dependent segregation of membrane proteins to distinct intracellular organelles. The dense granule localization of the single pass transmembrane protein GRA4 could be completely rerouted to the Golgi and cell surface simply by replacement of its TMD with that from either vesicular stomatitis virus G or the low density lipoprotein (LDL) receptor. Mutational and biochemical analyses suggested that this effect was not caused by any specific sequence motif or strength of membrane association of the GRA4 TMD. Instead, a property imparted by the vesicular stomatitis virus G or LDL receptor TMDs, both of which are longer than the GRA4 TMD, appeared to be a decisive factor. Indeed, shortening the LDL receptor TMD to a length similar to that of GRA4 resulted in dense granule localization, whereas lengthening the GRA4 TMD resulted in rerouting to the Golgi. From these data, we conclude that although the TMD may not necessarily be a sole determinant in membrane protein sorting, its properties can markedly modulate the utilization of more conventional signal-mediated sorting pathways.     

Transmembrane Ca2+ gradient and function of membrane proteins

This review will focus on the recent advance in the study of effect of transmembrane Ca²⁺ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca²⁺ gradient and sarcoplasmic reticulum Ca²⁺-ATPase; 2. Effect of transmembrane Ca²⁺ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca²⁺ gradient may play an important role in modulating the conformation and activity of SR Ca²⁺-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Ca_i Ca²⁺ inside vesicles - Ca₀ Ca²⁺ outside vesicles - SR sarcoplasmic reticulum - PC phosphatidylcholine - PS phosphatidylserine - PG phosphatidylglycerol - PE phosphatidylethanolamine - DPH 1,6-diphenyl-1,3,5-hexatriene - n-AS n-(9-anthroyloxy) fatty acids - TMA-DPH 1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - -AR -adrenergic receptors - DHA dihydroalprenolol - AC adenylate cyclase - AC·Lca+– higher Ca²⁺ inside vesicles - AC·Lca– – lower Ca²⁺ on both side of vesicles - AC·Lca++ higher Ca²⁺ on both side of vesicles - AC·Lca– + higher Ca²⁺ outside vesicles - cAMP cyclic adenosine monophosphate - Gs stimulatory GTP-binding protein - GTP guanosine triposphate - GTPS guanosine 50-(3-thiotriphosphate)     

The role of tryptophan side chains in membrane protein anchoring and hydrophobic mismatch

Tryptophan (Trp) is abundant in membrane proteins, preferentially residing near the lipid–water interface where it is thought to play a significant anchoring role. Using a total of 3 μs of molecular dynamics simulations for a library of hydrophobic WALP-like peptides, a long poly-Leu α-helix, and the methyl-indole analog, we explore the thermodynamics of the Trp movement in membranes that governs the stability and orientation of transmembrane protein segments. We examine the dominant hydrogen-bonding interactions between the Trp and lipid carbonyl and phosphate moieties, cation–π interactions to lipid choline moieties, and elucidate the contributions to the thermodynamics that serve to localize the Trp, by ~ 4 kcal/mol, near the membrane glycerol backbone region. We show a striking similarity between the free energy to move an isolated Trp side chain to that found from a wide range of WALP peptides, suggesting that the location of this side chain is nearly independent of the host transmembrane segment. Our calculations provide quantitative measures that explain Trp's role as a modulator of responses to hydrophobic mismatch, providing a deeper understanding of how lipid composition may control a range of membrane active peptides and proteins.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast.

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

Discontinuous membrane helices in transport proteins and their correlation with function

Alpha-helical bundles and beta-barrel proteins represent the two basic types of architecture known for integral membrane proteins. Irregular structural motifs have been revealed with the growing number of structures determined. "Discontinuous" helices are present in membrane proteins that actively transport ions. In the Ca(2+)-ATPase, a primary active transporter, and in the secondary transporters NhaA, LeuT(Aa), ClC H(+)/Cl(-) exchanger and Glt(Ph), the helical structure of two membrane segments is interrupted and the interjacent polypeptide chain forms an extended peptide. The discontinuous helices are integrated in the membrane either as transmembrane-spanning or hairpin-type segments. In addition, the secondary transporters have inverted internal duplication domains, which are only weakly correlated with their amino acid sequence. The symmetry comprises either parts of or the complete molecule, but always includes the discontinuous helices. The helix-peptide-helix motif is correlated with the ion translocation function. The extended peptides with their backbone atoms, the helix termini and the polar/charged amino acid residues in close vicinity provide the basis for ion recognition, binding and translocation.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

Quantitative analysis of membrane distortions induced by mismatch of protein and lipid hydrophobic thickness

The phase transitional behaviour of bilayers of the phospholipid l--ditridecanoylphos-phatidylcholine is studied as a function of protein content for the reaction center (RC) and an antenna protein (LHCP) of the bacterial photosynthetic apparatus. As membrane and protein are structurally well characterized the experimental results can be quantitatively compared with those of calculations based upon elastic models within the Landaude Gennes-theory. Agreement between theory and experiment demonstrates that dominant elastic forces result from a mismatch of hydrophobic regions of membrane and protein. The data also indicate that RC are present in a monomeric form and LHCP in a highly aggregated form. In addition, the latter protein responds to changes in the lipid environment.     

Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.