Effect of detergents on the association of the glycophorin a transmembrane helix

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van't Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.     

Changes in apparent free energy of helix-helix dimerization in a biological membrane due to point mutations

We present an implementation of the TOXCAT membrane protein self-association assay that measures the change in apparent free energy of transmembrane helix dimerization caused by point mutations. Quantifying the reporter gene expression from cells carrying wild-type and mutant constructs shows that single point mutations that disrupt dimerization of the transmembrane domain of glycophorin A reproducibly lower the TOXCAT signal more than 100-fold. Replicate cultures can show up to threefold changes in the level of expression of the membrane bound fusion construct, and correcting for these variations improves the precision of the calculated apparent free energy change. The remarkably good agreement between our TOXCAT apparent free energy scale and free energy differences from sedimentation equilibrium studies for point mutants of the glycophorin A transmembrane domain dimer indicate that sequence changes usually affect membrane helix-helix interactions quite similarly in these two very different environments. However, the effects of point mutations at threonine 87 suggest that intermonomer polar contacts by this side-chain contribute significantly to dimer stability in membranes but not in detergents. Our findings demonstrate that a comparison of quantitative measurements of helix-helix interactions in biological membranes and genuine thermodynamic data from biophysical measurements on purified proteins can elucidate how changes in the lipidic environment modulate membrane protein stability.     

Detergent Properties Influence the Stability of the Glycophorin A Transmembrane Helix Dimer in Lysophosphatidylcholine Micelles

Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important.     

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins.     

The stability of transmembrane helix interactions measured in a biological membrane

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.     

Sequence-Specific Dimerization of a Transmembrane Helix in Amphipol A8-35

As traditional detergents might destabilize or even denature membrane proteins, amphiphilic polymers have moved into the focus of membrane-protein research in recent years. Thus far, Amphipols are the best studied amphiphilic copolymers, having a hydrophilic backbone with short hydrophobic chains. However, since stabilizing as well as destabilizing effects of the Amphipol belt on the structure of membrane proteins have been described, we systematically analyze the impact of the most commonly used Amphipol A8-35 on the structure and stability of a well-defined transmembrane protein model, the glycophorin A transmembrane helix dimer. Amphipols are not able to directly extract proteins from their native membranes, and detergents are typically replaced by Amphipols only after protein extraction from membranes. As Amphipols form mixed micelles with detergents, a better understanding of Amphipol-detergent interactions is required. Therefore, we analyze the interaction of A8-35 with the anionic detergent sodium dodecyl sulfate and describe the impact of the mixed-micelle-like system on the stability of a transmembrane helix dimer. As A8-35 may highly stabilize and thereby rigidify a transmembrane protein structure, modest destabilization by controlled addition of detergents and formation of mixed micellar systems might be helpful to preserve the function of a membrane protein in Amphipol environments.     

Mutational analysis of synaptobrevin transmembrane domain oligomerization

Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25). Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain. However, these reports disagree about the magnitude of dimerization, which makes it difficult to assess the biological relevance of this interaction. We used SDS-PAGE and the TOXCAT genetic assay to reexamine the homodimerization of the synaptobrevin transmembrane domain in detergents and the Escherichia coli inner membrane, respectively. To gauge the magnitude of synaptobrevin homodimerization, we used the well-characterized glycophorin A homodimer as a positive standard. In contrast to previous studies, we found synaptobrevin homodimerization in E. coli is very weak when compared to glycophorin A. Recombinant synaptobrevin forms a small amount of dimer and higher order oligomers in detergents that are highly dependent on solublization conditions. We estimate a dissociation constant of 10 mM for synaptobrevin dimerization in detergent. Thus, the dimerization of synaptobrevin in membranes is very weak, questioning any possible functional role for this association in vivo.     

Sequence specificity in the dimerization of transmembrane alpha-helices.

While several reports have suggested a role for helix-helix interactions in membrane protein oligomerization, there are few direct biochemical data bearing on this subject. Here, using mutational analysis, we show that dimerization of the transmembrane alpha-helix of glycophorin A in a detergent environment is spontaneous and highly specific. Very subtle changes in the side-chain structure at certain sensitive positions disrupt the helix-helix association. These sensitive positions occur at approximately every 3.9 residues along the helix, consistent with their comprising the interface of a closely fit transmembranous supercoil of alpha-helices. By contrast with other reported cases of interactions between transmembrane helices, the set of interfacial residues in this case contains no highly polar groups. Amino acids with aliphatic side chains define much of the interface, indicating that precise packing interactions between the helices may provide much of the energy for association. These data highlight the potential general importance of specific interactions between the hydrophobic anchors of integral membrane proteins.     

Synthetic peptides mimic the assembly of transmembrane glycoproteins

The composition of the intramembranous domains of many receptors are remarkably uniform, yet there is evidence that many transmembrane proteins associate together to form specific noncovalent homo- or heterocomplexes within the membrane. We have synthesized peptides corresponding to transmembrane domains of glycophorin A, glycophorin C, and the interleukin 2-receptor Tac antigen to study the interactions between transmembrane domains in vitro. Synthetic transmembrane glycophorin A peptide formed a complex with native glycophorin and glycoproteins of erythrocyte and K562 cell membranes that was reversible, specific, and could be demonstrated in a natural bilayer system in the absence of detergents. Synthetic glycophorin C and interleukin 2-receptor Tac antigen transmembrane peptides, although similar in amino acid composition, did not interact with glycophorin and did not inhibit the binding of the synthetic glycophorin A transmembrane peptide to native glycophorin. It is proposed that the transmembrane segments of receptor proteins contain not only the structural information necessary for insertion and anchoring but specific binding sites that mediate interactions between transmembrane glycoproteins.     

Polar/Ionizable Residues in Transmembrane Segments: Effects on Helix-Helix Packing

The vast majority of membrane proteins are anchored to biological membranes through hydrophobic α-helices. Sequence analysis of high-resolution membrane protein structures show that ionizable amino acid residues are present in transmembrane (TM) helices, often with a functional and/or structural role. Here, using as scaffold the hydrophobic TM domain of the model membrane protein glycophorin A (GpA), we address the consequences of replacing specific residues by ionizable amino acids on TM helix insertion and packing, both in detergent micelles and in biological membranes. Our findings demonstrate that ionizable residues are stably inserted in hydrophobic environments, and tolerated in the dimerization process when oriented toward the lipid face, emphasizing the complexity of protein-lipid interactions in biological membranes.     

Intermonomer Hydrogen Bonds Enhance GxxxG-Driven Dimerization of the BNIP3 Transmembrane Domain: Roles for Sequence Context in Helix-Helix Association in Membranes

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.     

Structural role of glycine in amyloid fibrils formed from transmembrane alpha-helices

Amyloid fibrils associated with diseases such as Alzheimer's are often derived from the transmembrane helices of membrane proteins. It is known that the fibrils have a cross-beta-sheet structure where main chain hydrogen bonding occurs between beta-strands in the direction of the fibril axis. However, the structural basis for how the membrane-spanning helix is converted into a beta-sheet or how protofibrils associate into fibrils is not known. Here, we use a model peptide corresponding to a portion of the single transmembrane helix of glycophorin A to investigate the structural role of glycine in amyloid-like fibrils formed from transmembrane helices. Glycophorin A contains a GxxxG motif that is found in many transmembrane sequences including that of the amyloid precursor protein and prion protein. We propose that glycine, which mediates helix interactions in membrane proteins, also provides key packing motifs when it occurs in beta-sheets. We show that glycines in the glycophorin A transmembrane helix promote extended beta-strand formation when the helix partitions into aqueous environments and stabilize the packing of beta-sheets in the formation of amyloid-like fibrils. We demonstrate that fibrillization can be disrupted with a new class of inhibitors that target the molecular grooves created by glycine.     

Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A

Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of β-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the C_α-C_β bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the β-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers.     

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix: a rapid way to map helix-helix interactions in integral membrane proteins.

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix-helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala-insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.     

Transmembrane helix–helix interactions are modulated by the sequence context and by lipid bilayer properties

Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix–helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix–helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix–helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Interhelical hydrogen bonding drives strong interactions in membrane proteins

Polar residues in transmembrane alpha-helices may strongly influence the folding or association of integral membrane proteins. To test whether a motif that promotes helix association in a soluble protein could do the same within a membrane, we designed a model transmembrane helix based on the GCN4 leucine zipper. We found in both detergent micelles and biological membranes that helix association is driven strongly by asparagine, independent of the rest of the hydrophobic leucine and/or valine sequence. Hydrogen bonding between membrane helices gives stronger associations than the packing of surfaces in glycophorin A helices, creating an opportunity to stabilize structures, but also implying a danger that non-specific interactions might occur. Thus, membrane proteins may fold to avoid exposure of strongly hydrogen bonding groups at their lipid exposed surfaces.     

Dimerization of the erythropoietin receptor transmembrane domain in micelles

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.     

Standardizing the free energy change of transmembrane helix-helix interactions

Side-to-side associations of transmembrane alpha-helices are integral components of the structure and function of helical membrane proteins. A fundamental unknown in the understanding of the chemical principles driving the lateral interactions between transmembrane alpha-helices is the balance of forces arising from the polypeptide sequence versus the hydrophobic solvent. To begin to address this question, a consideration of basic thermodynamic principles has been applied to assess the experimental free energy change associated with transmembrane helix dimerization in micelles. This analysis demonstrates the ability to partition the apparent free energy of transmembrane helix-helix association into two components. The first component is a statistical energy term, which arises from the fact that there are an unequal number of reactants and products. The second component is a standard state free energy change, which informs on the molecular details of the transmembrane helix self-association reaction. The advantage of separating these two energy terms arises from the fact that extrapolation to the standard state free energy change normalizes the statistical energy term so that it applies equivalently in all experimental systems. Accompanying experimental results for the glycophorin A transmembrane alpha-helix dimer measured in micelles are well described by these theoretical components assuming an ideal-dilute solution.