Role of hydrogen bonding and helix-lipid interactions in transmembrane helix association

To explore the role of hydrogen bonding and helix-lipid interactions in transmembrane helix association, we have calculated the potential of mean force (PMF) as a function of helix-helix distance between two pVNVV peptides, a transmembrane model peptide based on the GCN4 leucine-zipper, in a dimyristoylphosphatidylcholine (DMPC) membrane. The peptide name pVNVV represents the interfacial residues in the heptad repeat of the dimer. The free energy decomposition reveals that the total PMF consists of two competing contributions from helix-helix and helix-lipid interactions. The direct, favorable helix-helix interactions arise from the specific contribution from the helix-facing residues and the generic contribution from the lipid-facing residues. The Asn residues in the middle of the helices show the most significant per-residue contribution to the PMF with various hydrogen bonding patterns as a function of helix-helix distance. Release of lipid molecules between the helices into bulk lipid upon helix association makes the helix-lipid interaction enthalpically unfavorable but entropically favorable. Interestingly, the resulting unfavorable helix-lipid contribution to the PMF correlates well with the cavity volume between the helices. The calculated PMF with an Asn-to-Val mutant (pVNVV --> pVVVV) shows a dramatic free energy change upon the mutation, such that the mutant appears not to form a stable dimer below a certain peptide concentration, which is in good agreement with available experimental data of a peptide with the same heptad repeat. A transmembrane helix association mechanism and its implications in membrane protein folding are also discussed.     

beta-Barrel membrane bacterial proteins: structure, function, assembly and interaction with lipids

Membrane proteins, although constituting about one-third of all proteins encoded by the genomes of living organisms, are still strongly underrepresented in the database of 3D protein structures, which reflects the big challenge presented by this class of proteins. Structural biologists, by employing electron and x-ray approaches, are continuously revealing new and fundamental insights into the structure, function, assembly and interaction with lipids of membrane proteins. To date, two structural motifs, alpha-helices and beta-sheets, have been found in membrane proteins and interestingly these two structural motives correlate with the location: while alpha-helical bundles are most often found in the receptors and ion channels of plasma and endoplasmic reticulum membranes, beta-barrels are restricted to the outer membrane of Gram-negative bacteria and in the mitochondrial membrane, and represent the structural motif used by several microbial toxins to form cytotoxic transmembrane channels. The beta-barrel, while being a rigid and stable motif is a versatile scaffold, having a wide variation in the size of the barrel, in the mechanism to open or close the gate and to impose selectivity on substrates. Even if the number of x-ray structures of integral membrane proteins has greatly increased in recent years, only a few of them provide information at a molecular level on how proteins interact with lipids that surround them in the membrane. The detailed mechanism of protein lipid interactions is of fundamental importance for understanding membrane protein folding, membrane adsorption, insertion and function in lipid bilayers. Both specific and unspecific interactions with lipids may participate in protein folding and assembly.     

Mechanisms of the interaction of alpha-helical transmembrane peptides with phospholipid bilayers

The synthetic peptide acetyl-K(2)-G-L(24)-K(2)-A-amide (P(24)) and its analogs have been successfully utilized as models of the hydrophobic transmembrane alpha-helical segments of integral membrane proteins. The central polyleucine region of these peptides was designed to form a maximally stable, very hydrophobic alpha-helix which will partition strongly into the hydrophobic environment of the lipid bilayer core, while the dilysine caps were designed to anchor the ends of these peptides to the polar surface of the lipid bilayer and to inhibit the lateral aggregation of these peptides. Moreover, the normally positively charged N-terminus and the negatively charged C-terminus have both been blocked in order to provide a symmetrical tetracationic peptide, which will more faithfully mimic the transbilayer region of natural membrane proteins and preclude favorable electrostatic interactions. In fact, P(24) adopts a very stable alpha-helical conformation and transbilayer orientation in lipid model membranes. The results of our recent studies of the interaction of this family of alpha-helical transmembrane peptides with phospholipid bilayers are summarized here.     

Insertion and assembly of membrane proteins via simulation

Interactions of lipids are central to the folding and stability of membrane proteins. Coarse-grained molecular dynamics simulations have been used to reveal the mechanisms of self-assembly of protein/membrane and protein/detergent complexes for representatives of two classes of membrane protein, namely, glycophorin (a simple alpha-helical bundle) and OmpA (a beta-barrel). The accuracy of the coarse-grained simulations is established via comparison with the equivalent atomistic simulations of self-assembly of protein/detergent micelles. The simulation of OmpA/bilayer self-assembly reveals how a folded outer membrane protein can be inserted in a bilayer. The glycophorin/bilayer simulation supports the two-state model of membrane folding, in which transmembrane helix insertion precedes dimer self-assembly within a bilayer. The simulations also suggest that a dynamic equilibrium exists between the glycophorin helix monomer and dimer within a bilayer. The simulated glycophorin helix dimer is remarkably close in structure to that revealed by NMR. Thus, coarse-grained methods may help to define mechanisms of membrane protein (re)folding and will prove suitable for simulation of larger scale dynamic rearrangements of biological membranes.     

Dramatic destabilization of transmembrane helix interactions by features of natural membrane environments

Membrane proteins have evolved to fold and function in a lipid bilayer, so it is generally assumed that their stability should be optimized in a natural membrane environment. Yet optimal stability is not always in accord with optimization of function, so evolutionary pressure, occurring in a complex membrane environment, may favor marginal stability. Here, we find that the transmembrane helix dimer, glycophorin A (GpATM), is actually much less stable in the heterogeneous environment of a natural membrane than it is in model membranes and even common detergents. The primary destabilizing factors are electrostatic interactions between charged lipids and charged GpATM side chains, and nonspecific competition from other membrane proteins. These effects overwhelm stabilizing contributions from lateral packing pressure and excluded volume. Our work illustrates how evolution can employ membrane composition to modulate protein stability.     

Strength of Calpha-H...O=C hydrogen bonds in transmembrane proteins

A large number of Calpha-H...O contacts are present in transmembrane protein structures, but contribution of such interactions to protein stability is still not well understood. According to previous ab initio quantum calculations, the stabilization energy of a Calpha-H...O contact is about 2-3 kcal/mol. However, experimental studies on two different Calpha-H...O hydrogen bonds present in transmembrane proteins lead to conclusions that one contact is only weakly stabilizing and the other is not even stabilizing. We note that most previous computational studies were on optimized geometries of isolated molecules, but the experimental measurements were on those in the structural context of transmembrane proteins. In the present study, 263 Calpha-H...O=C contacts in alpha-helical transmembrane proteins were extracted from X-ray crystal structures, and interaction energies were calculated with quantum mechanical methods. The average stabilization energy of a Calpha-H...O=C interaction was computed to be 1.4 kcal/mol. About 13% of contacts were stabilizing by more than 3 kcal/mol, and about 11% were destabilizing. Analysis of the relationships between energy and structure revealed four interaction patterns: three types of attractive cases in which additional Calpha-H...O or N-H...O contact is present and a type of repulsive case in which repulsion between two carbonyl oxygen atoms occur. Contribution of Calpha-H...O=C contacts to protein stability is roughly estimated to be greater than 5 kcal/mol per helix pair for about 16% of transmembrane helices but for only 3% of soluble protein helices. The contribution would be larger if Calpha-H...O contacts involving side chain oxygen were also considered.     

Anomalous diffusion of oligomerized transmembrane proteins

Transmembrane proteins frequently form (transient) oligomers on biomembranes, e.g., while participating in protein sorting and signaling events. Using coarse-grained membrane simulations we show here that transmembrane proteins show a subdiffusive motion on short time scales when being part of a linear oligomer, i.e., a flexible polymer, embedded in a two-dimensional membrane. Our results are in agreement with previous experimental observations. They further indicate that polymers of transmembrane proteins are well described by predictions from Rouse theory in two dimensions even in the presence of hydrodynamic interactions.     

De novo design and molecular assembly of a transmembrane diporphyrin-binding protein complex

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.     

Lipid/protein interactions and the membrane/water interfacial region

Despite the importance of lipid/protein interactions in the folding, assembly, stability, and function of membrane proteins, information at an atomic level on how such proteins interact with the lipids that surround them remains sparse. The dynamics and flexible nature of the protein/bilayer interaction make it difficult to study, for example, by crystallographic means. However, based on recent progress in molecular simulations of membranes it is possible to address this problem computationally. This communication reports one of the first attempts to use multiple ns molecular simulations to establish a qualitative picture of the intermolecular interactions between the lipids of a bilayer and two topologically different membrane proteins for which a high resolution (2 A or better) X-ray structure is available.     

Folding kinetics of a naturally occurring helical peptide: implication of the folding speed limit of helical proteins

The folding mechanism and dynamics of a helical protein may strongly depend on how quickly its constituent alpha-helices can fold independently. Thus, our understanding of the protein folding problem may be greatly enhanced by a systematic survey of the folding rates of individual alpha-helical segments derived from their parent proteins. As a first step, we have studied the relaxation kinetics of the central helix (L9:41-74) of the ribosomal protein L9 from the bacterium Bacillus stearothermophilus , in response to a temperature-jump ( T-jump) using infrared spectroscopy. L9:41-74 has been shown to exhibit unusually high helicity in aqueous solution due to a series of side chain-side chain interactions, most of which are electrostatic in nature, while still remaining monomeric over a wide concentration range. Thus, this peptide represents an excellent model system not only for examining how the folding rate of naturally occurring helices differs from that of the widely studied alanine-based peptides, but also for estimating the folding speed limit of (small) helical proteins. Our results show that the T-jump induced relaxation rate of L9:41-74 is significantly slower than that of alanine-based peptides. For example, at 11 degrees C its relaxation time constant is about 2 micros, roughly seven times slower than that of SPE(5), an alanine-rich peptide of similar chain length. In addition, our results show that the folding rate of a truncated version of L9:41-74 is even slower. Taken together, these results suggest that individual alpha-helical segments in proteins may fold on a time scale that is significantly slower than the folding time of alanine-based peptides. Furthermore, we argue that the relaxation rate of L9:41-74 measured between 8 and 45 degrees C provides a realistic estimate of the ultimate folding rate of (small) helical proteins over this temperature range.     

Single particle tracking reveals corralling of a transmembrane protein in a double-cushioned lipid bilayer assembly

A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a “single-cushion” POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains ( 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC?PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.     

Noncovalent keystone interactions controlling biomembrane structure

There is a biomedical need to develop molecular recognition systems that selectively target the interfaces of protein and lipid aggregates in biomembranes. This is an extremely challenging problem in supramolecular chemistry because the biological membrane is a complex dynamic assembly of multifarious molecular components with local inhomogeneity. Two simplifying concepts are presented as a framework for basing molecular design strategies. The first generalization is that association of two binding partners in a biomembrane will be dominated by one type of non-covalent interaction which is referred to as the keystone interaction. Structural mutations in membrane proteins that alter the strength of this keystone interaction will likely have a major effect on biological activity and often will be associated with disease. The second generalization is to view the structure of a cell membrane as three spatial regions, that is, the polar membrane surface, the midpolar interfacial region and the non-polar membrane interior. Each region has a distinct dielectric, and the dominating keystone interaction between binding partners will be different. At the highly polar membrane surface, the keystone interactions between charged binding partners are ion-ion and ion-dipole interactions; whereas, ion-dipole and ionic hydrogen bonding are very influential at the mid-polar interfacial region. In the non-polar membrane interior, van der Waals forces and neutral hydrogen bonding are the keystone interactions that often drive molecular association. Selected examples of lipid and transmembrane protein association systems are described to illustrate how the association thermodynamics and kinetics are dominated by these keystone noncovalent interactions.     

beta-Barrel transmembrane proteins: Geometric modelling, detection of transmembrane region, and structural properties

The location of the membrane lipid bilayer relative to a transmembrane protein structure is important in protein engineering. Since it is not present on the determined structures, it is essential to automatically define the membrane embedded protein region in order to test mutation effects or to design potential drugs. beta-Barrel transmembrane proteins, present in nature as outer membrane proteins (OMPs), comprise one of the two transmembrane protein fold classes. Lately, the number of their determined structures has increased and this enables the implementation and evaluation of structure-based annotation methods and their more comprehensive study. In this paper, we propose two new algorithms for (i) the geometric modelling of beta-barrels and (ii) the detection of the transmembrane region of a beta-barrel transmembrane protein. The geometric modelling algorithm combines a non-linear least square minimization method and a genetic algorithm in order to find the characteristics (axis, radius) of a shape with axial symmetry which best models a beta-barrel. The transmembrane region is detected by profiling the external residues of the beta-barrel along its axis in terms of hydrophobicity and existence of aromatic and charged residues. TbB-Tool implements these algorithms and is available in . A non-redundant set of 22 OMPs is used in order to evaluate the algorithms implemented and the results are very satisfying. In addition, we quantify the abundance of all amino acids and the average hydrophobicity for external and internal beta-stranded residues along the axis of beta-barrel, thus confirming and extending other researchers' results.     

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.     

Quantification of protein-protein interactions within membranes by fluorescence correlation spectroscopy

The characterization of interactions between membrane proteins as they take place within the lipid bilayer poses a technical challenge, which is currently very difficult and, in many cases, impossible to overcome. The recent development of a method based in the combination two-color fluorescence cross-correlation spectroscopy with scanning of the focal volume allows the detection and quantification of interactions between biomolecules inserted in biological membranes. This powerful strategy has allowed the quantitative analysis of diverse systems, such as the association between proteins of the Bcl-2 family involved in apoptosis regulation or the binding between a growth factor and its receptor during signaling. Here, we review the last developments to quantify protein/protein interactions in lipid membranes and focus on the use of fluorescence-correlation-spectroscopy approaches for that purpose.     

Peptide-lipid interactions: insights and perspectives

As the number of membrane proteins in the Protein Data Bank increases, efforts to understand how they interact with their natural environment are increasing in importance. A number of membrane proteins crystallise with lipid molecules implicitly bound at discrete locations that are consistent with the transmembrane regions of the protein. Bioinformatics studies also point to the specific interactions of some amino acids with membrane lipids. The results of experiments using model systems are revealing how these interactions contribute to the stability of both the protein and the membrane in which it is embedded. From a different perspective, the processes involved in the binding of peptides to membrane surfaces to produce a variety of effects are being understood in ever-increasing detail. This review describes current research efforts and thinking in this area.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

Engineering a beta-sheet protein toward the folding speed limit

Recent experimental studies have shown that alpha-helical proteins can approach the folding "speed limit", where folding switches from an activated to a downhill process in free energy. beta-sheet proteins are generally thought to fold more slowly than helix bundles. However, based on studies of hairpins, folding should still be able to approach the microsecond time scale. Here we demonstrate how the hPin1 WW domain, a triple-stranded beta-sheet protein with a sharp thermodynamic melting transition, can be engineered toward the folding "speed limit" without a significant loss in thermal denaturation cooperativity.