Mechanistic insights from functional characterization of an unnatural His37 mutant of the influenza A/M2 protein.

The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH8 in bilayers and is virtually inactive at external pH7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Nanosecond Dynamics of InfluenzaA/M2TM and an Amantadine Resistant Mutant Probed by Time-Dependent Red Shifts of a Native Tryptophan.

Proteins involved in functions such as electron transfer or ion transport must be capable of stabilizing transient charged species on time scales ranging from picoseconds to microseconds. We study the influenza A M2 proton channel, containing a tryptophan residue that serves as an essential part of the proton conduction pathway. We induce a transition dipole in tryptophan by photoexcitation, and then probe the dielectric stabilization of its excited state. The magnitude of the stabilization over this time regime was larger than that generally found for tryptophan in membrane or protein environments. M2 achieves a water-like stabilization over a 25 nanosecond time scale, slower than that of bulk water, but sufficiently rapid to contribute to stabilization of charge as protons diffuse through the channel. These measurements should stimulate future MD studies to clarify the role of sidechain versus non-bulk water in defining the process of relaxation.

Use of thiol-disulfide exchange method to study transmembrane peptide association in membrane environments.

The development of methods for reversibly folding membrane proteins in a two-state manner remains a considerable challenge for studies of membrane protein stability. In recent years, a variety of techniques have been established and studies of membrane protein folding thermodynamics in the native bilayer environments have become feasible. Here we present the thiol-disulfide exchange method, a promising experimental approach for investigating the thermodynamics of transmembrane (TM) helix-helix association in membrane-mimicking environments. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. This experimental method provides a broadly applicable tool for thermodynamic studies of folding, oligomerization, and helix-helix interactions of membrane proteins.

The interplay of functional tuning, drug resistance, and thermodynamic stability in the evolution of the M2 proton channel from the influenza A virus.

We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2's specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.

Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer.

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG(79)VMAGVIGTILLISWG(94)IKK was labeled at G(79) and G(94) with (13)C=(16)O or (13)C=(18)O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either (13)C=(16)O or (13)C=(18)O substitution and a heterodimer when one had (13)C=(16)O substitution and the other had (13)C=(18)O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3-6.3 cm(-1) arises from through-space interactions between amide units on different helices. The angle between the two Gly(79) amide-I transition dipoles, estimated at 103 degrees from linear IR spectroscopy and 110 degrees from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45 degrees. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution.

Truncation of a cross-linked GCN4-p1 coiled coil leads to ultrafast folding.

Structural perturbation has been extensively used in protein folding studies because it yields valuable conformational information regarding the folding process. Here we have used N-terminal truncation on a cross-linked variant of the GCN4-p1 leucine zipper, aiming to develop a better understanding of the folding mechanism of the coiled-coil motif. Our results indicate that removing the first heptad repeat in this cross-linked GCN4-p1 coiled coil significantly decreases the folding free energy barrier and results in a maximum folding rate of (2.0 +/- 0.3 micros)(-1), which is approximately 50 times faster than that of the full-length protein. Therefore, these results suggest that a set of native or nativelike tertiary interactions, distributed throughout the entire sequence, collectively stabilize the folding transition state of the GCN4-p1 coiled coil. While stable subdomains or triggering sequences have been shown to be critical to the stability of GCN4 coiled coils, our results suggest that the folding of such a subdomain does not seem to dictate the overall folding kinetics.

Characterization of a membrane protein folding motif, the Ser zipper, using designed peptides.

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.

Synergistic interactions between aqueous and membrane domains of a designed protein determine its fold and stability.

Membrane-spanning proteins contain both aqueous and membrane-spanning regions, both of which contribute to folding and stability. To explore the interplay between these two domains we have designed and studied the assembly of coiled-coil peptides that span from the membrane into the aqueous phase. The membrane-spanning segment is based on MS1, a transmembrane coiled coil that contains a single Asn at a buried a position of a central heptad in its sequence. This Asn has been shown to drive assembly of the monomeric peptide in a membrane environment to a mixture of dimers and trimers. The coiled coil has now been extended into the aqueous phase by addition of water-soluble helical extensions. Although too short to fold in isolation, these helical extensions were expected to interact synergistically with the transmembrane domain and modulate its stability as well as its conformational specificity for forming dimers versus trimers. One design contains Asn at a position of the aqueous helical extension, which was expected to specify a dimeric state; a second peptide, which contains Val at this position, was expected to form trimers. The thermodynamics of assembly of the hybrid peptides were studied in micelles by sedimentation equilibrium ultracentrifugation. The aqueous helical extensions indeed conferred additional stability and conformational specificity to MS1 in the expected manner. These studies highlight the delicate interplay between membrane-spanning and water-soluble regions of proteins, and demonstrate how these different environments define the thermodynamics of a given specific interaction. In this case, an Asn in the transmembrane domain provided a strong driving force for folding but failed to specify a unique oligomerization state, while an Asn in the water-soluble domain was able to define specificity for a specific aggregation state as well as modulate stability.

Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers.

Despite significant efforts and promising progress, the understanding of membrane protein folding lags behind that of soluble proteins. Insights into the energetics of membrane protein folding have been gained from biophysical studies in membrane-mimicking environments (primarily detergent micelles). However, the development of techniques for studying the thermodynamics of folding in phospholipid bilayers remains a considerable challenge. We had previously used thiol-disulfide exchange to study the thermodynamics of association of transmembrane alpha-helices in detergent micelles; here, we extend this methodology to phospholipid bilayers. The system for this study is the homotetrameric M2 proton channel protein from the influenza A virus. Transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. Thiol-disulfide exchange under equilibrium conditions was used to quantitatively measure the thermodynamics of this folding interaction in phospholipid bilayers. The effects of phospholipid acyl chain length and cholesterol on the peptide association were investigated. The association of the helices strongly depends on the thickness of the bilayer and cholesterol levels present in the phospholipid bilayer. The most favorable folding occurred when there was a good match between the width of the apolar region of the bilayer and the hydrophobic length of the transmembrane helix. Physiologically relevant variations in the cholesterol level are sufficient to strongly influence the association. Evaluation of the energetics of peptide association in the presence and absence of cholesterol showed a significantly tighter association upon inclusion of cholesterol in the lipid bilayers.

Mimicking photosynthesis in a computationally designed synthetic metalloprotein.

While advances in protein design have made possible the construction of protein architectures with nativelike properties and predictable structures and function, there are as of yet no examples of functional, protein-based, solar energy conversion systems. This communication describes the design and characterization of an artificial reaction center (RC) protein that closely resembles the function of the natural photosynthetic RC. The synthetic protein, designed by the protein design program CORE, participates in multiple reduction/oxidation cycles with exogenous acceptors/donors following photoexcitation. The designed metalloprotein, aRC, consists of a tetrahelical bundle functionalized with two bis-histidine bound metal cofactors: a Ru(bpy)2 moiety and a heme group. Two distinct bis-histidine binding sites were engineered for each of these metal centers. Photoexcitation of aRC results in rapid ET from the RuII complex to the heme group (kET >/= 5 x 1010 s-1) yielding a long-lived (70 ns) charge-separated state (CSS), RuIII/FeII. This long-lived CSS participates in subsequent ET reactions with exogenous donors and acceptors in multiple photocycles, thus mimicking the basic function of native photosynthetic RCs. This study illustrates the successful design and construction of a protein-based functional charge separation device using a combination of automated computational protein design and knowledge of the engineering principles of biological electron tunneling extracted from natural electron-transfer systems. To our knowledge, this represents the first example of a functional protein-based artificial reaction center.

Determination of membrane protein stability via thermodynamic coupling of folding to thiol-disulfide interchange.

Although progress has been made in understanding the thermodynamic stability of water-soluble proteins, our understanding of the folding of membrane proteins is at a relatively primitive level. A major obstacle to understanding the folding of membrane proteins is the discovery of systems in which the folding is in thermodynamic equilibrium, and the development of methods to quantitatively assess this equilibrium in micelles and bilayers. Here, we describe the application of disulfide cross-linking to quantitatively measure the thermodynamics of membrane protein association in detergent micelles. The method involves initiating disulfide cross-linking of a protein under reversible redox conditions in a thiol-disulfide buffer and quantitative assessment of the extent of cross-linking at equilibrium. The 19-46 alpha-helical transmembrane segment of the M2 protein from the influenza A virus was used as a model membrane protein system for this study. Previously it has been shown that transmembrane peptides from this protein specifically self-assemble into tetramers that retain the ability to bind to the drug amantadine. We used thiol-disulfide exchange to quantitatively measure the tetramerization equilibrium of this transmembrane protein in dodecylphosphocholine (DPC) detergent micelles. The association constants obtained agree remarkably well with those derived from analytical ultracentrifugation studies. The experimental method established herein should provide a broadly applicable tool for thermodynamic studies of folding, oligomerization and protein-protein interactions of membrane proteins.