Helix Dimer Research Articles

A FlAsH–Tetracysteine Assay for Quantifying the Association and Orientation of Transmembrane α‐Helices

Lighting up protein dimers in membranes: The association of membrane proteins plays a critical role in biology. We describe a FlAsH–tetracysteine (tC) assay that detects protein dimerization in lipid bilayers with a fluorescent readout. The stringent spatial requirement of FlAsH–tC complex formation readily differentiates helix dimers in parallel and antiparallel orientations. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms [1]. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. In all bilayers, sequence-specificity is evident by the formation of a clear interface between the helices that is modulated by the lipid environment. Extracted estimates of the dimerization affinity are in excellent agreement with experimental data [2]. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces. We present results that support the role of lipid-mediated contributions to such effects with major implications on protein function. [1] Janosi L. and Doxastakis M., “Accelerating flat-histogram methods for potential of mean force calculations”, J. Chem. Phys., 131, 054105 (2009) [2] Janosi L., Prakash A. and Doxastakis M., “Lipid-Modulated sequence-specific association of Glycophorin A in membranes”, Biophys. J., 99, 284-292 (2010)

Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix–helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys–Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1–E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.

Effect of polarization on the stability of a helix dimer

Molecular dynamics (MD) simulations have been carried out to study helix–helix interaction using both standard AMBER and polarized force fields. Comparison of the two simulations shows that electrostatic polarization of intra-protein hydrogen bonds plays a significant role in stabilizing the structure of helix dimer. This stabilizing effect is clearly demonstrated by examining the monomer structure, helix crossing angle and stability of backbone hydrogen bonds under AMBER and PPC. Since reliable prediction of protein–protein structure is a significant challenge, the current study should help shed light on the importance of electrostatic polarization of protein in helix–helix interaction and helix bundle structures.

Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane α-helical dimers: ephrin receptor A1

Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide–peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix–helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active ‘peptide interceptors’ specifically targeting membrane domains of proteins.

Unfolding a transmembrane helix dimer: A FRET study in mixed micelles

The exact nature of membrane protein folding and assembly is not understood in detail yet. Addition of SDS to a membrane protein dissolved in mild, non-polar detergent results in formation of mixed micelles and in subsequent denaturation of higher ordered membrane protein structures. The exact nature of this denaturation event is, however, enigmatic, and separation of an individual helix pair in mixed micelles has also not been reported yet. Here we followed unfolding of the human glycophorin A transmembrane helix dimer in mixed micelles by fluorescence spectroscopy. Energy transfer between differently labelled glycophorin A transmembrane helices decreased with increasing SDS mole fractions albeit without modifying the helicity of the peptides. The energetics and kinetics of the dimer dissociation in mixed micelles is analyzed and discussed, and the experimental data demonstrate that mixed micelles can be used as a general method to investigate unfolding of alpha-helical membrane proteins.

Molecular Dynamics Simulations of the Dimerization of Transmembrane α-Helices

The lateral association of transmembrane (TM) α-helices within a lipid bilayer environment is a key stage in the folding of membrane proteins. It may also play a role in signalling across cell membranes. Dimerization of TM helices provides a simple example of such lateral association. Direct atomistic (AT) resolution MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations. We demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multi-scale approach for generating and refining models of TM helix dimers. This approach has been applied to a number of examples, including the glycophorin TM helix dimer (a paradigm for helix/helix packing) [1], and the TM domain of the syndecan-2 receptor protein, which contains a GxxxG motif comparable to that of glycophorin. The multi-scale approach has also been applied to a more complex system, the heterodimeric αIIb/β3 integrin TM helix dimer.[1] Psachoulia, E., P. J. Bond, P. W. Fowler, and M. S. P. Sansom. 2008. Helix-helix interactions in membrane proteins: coarse grained simulations of glycophorin helix dimerization. Biochem. 47:10503-105012.

Involvement of Transmembrane Helix Dimerization and Rotation in Signaling by the Thrombopoietin Receptor

In this study, we investigate the role of the transmembrane (TM) domain in the activation of one member of the cytokine receptor family: the thrombopoietin receptor (TpoR). The TM domain is thought to play a key role in the activation by facilitating receptor homodimerization and by transmitting the ligand-induced re-orientation of the extracellular domain to the cytoplasmic domain through conformational changes. However, the precise mechanisms underlying these events are not fully understood. Here, we considered several unanswered questions: Is the homodimerization of TpoR TM domain important in receptor activation? Which TM residues are involved in stabilizing receptor interactions? Additionally, we investigated the role of a constitutively activating mutation (S505N) and the mechanism of action of a piperidine-4-carboxylic acid TpoR agonist (Compound 2). We show that the TM domain of the human TpoR dimerizes strongly and that the full-length receptors exist as homodimers on the surface of mammalian cells in the absence of ligand. Our results indicate that TpoR can adopt two different conformations involving two different sets of residues. One of the contact interfaces mimics an inactive unliganded TpoR dimer and the other corresponds to an active conformation that is compatible with the constitutive signaling induced by the S505N mutation. We also show that Compound 2 interacts specifically with the N-terminus portion of the active dimer conformation. Overall, our results give new insight into the role of the TM domain in the activation mechanism of TpoR and provide a more detailed model of cytokine receptor activation and modulation by small molecule agonists.

Molecular Dynamics Simulations of the Dimerization of Transmembrane α-Helices

Membrane proteins account for nearly a quarter of all genes, but their structure and function remain incompletely understood. Most membrane proteins have transmembrane (TM) domains made up of bundles of hydrophobic alpha-helices. The lateral association of TM helices within the lipid bilayer is a key stage in the folding of membrane proteins. It may also play a role in signaling across cell membranes. Dimerization of TM helices is a simple example of such lateral association. Molecular dynamics (MD) simulations have been used for over a decade to study membrane proteins in a lipid bilayer environment. However, direct atomistic (AT) MD simulation of self-assembly of a TM helix bundle remains challenging. AT-MD may be complemented by coarse-grained (CG) simulations, in which small numbers of atoms are grouped together into particles. In this Account, we demonstrate how CG-MD may be used to simulate formation of dimers of TM helices. We also show how a serial combination of CG and AT simulation provides a multiscale approach for generating and refining models of TM helix dimers. The glycophorin A (GpA) TM helix dimer represents a paradigm for helix-helix packing, mediated by a GxxxG sequence motif. It is well characterized experimentally and so is a good test case for evaluating computational methods. CG-MD simulations in which two separate TM helices are inserted in a lipid bilayer result in spontaneous formation of a right-handed GpA dimer, in agreement with NMR structures. CG-MD models were evaluated via comparison with data on destabilizing mutants of GpA. Such mutants increased the conformational flexibility and the dissociation constants of helix dimers. GpA dimers have been used to evaluate a multiscale approach: A CG model is converted to an AT model, which is used as the basis of an AT-MD simulation. Comparison of three AT-MD simulations of GpA, one starting from a CG model and two starting from NMR structures, leads to convergence to a common refined structure for the dimer. CG-MD self-assembly has also been used to model dimerization of the TM domain of the syndecan-2 receptor protein. This TM helix contains a GxxxG motif, which mediates right-handed helix packing comparable to that of the GxxxG motif in GpA. The multiscale approach has been applied to a more complex system, the heterodimeric alphaIIb/beta3 integrin TM helix dimer. In CG-MD, both right-handed and left-handed structures were formed. Subsequent AT-MD simulations showed that the right-handed structure was more stable, yielding a dimer in which the GxxxG motif of the alphaIIb TM helix packed against a hydrophobic surface of the beta3 helix in a manner comparable to that observed in two recent NMR studies. This work demonstrates that the multiscale simulation approach can be used to model simple membrane proteins. The method may be applied to more complex proteins, such as the influenza M2 channel protein. Future refinements, such as extending the multiscale approach to a wider range of scales (from CG through QM/MM simulations, for example), will expand the range of applications and the accuracy of the resultant models.

The Transmembrane Structure of Integrin αIIbβ3: Significance for Signal Transduction

The formation of multicellular organisms requires concerted action by cells, which alter their adhesive and migratory behaviors. Cell adhesion and migration are tightly regulated by intraand extracellular signals, which are conveyed through the cellular membrane by specialized receptors known as integrins. Integrins are the starting point of a variety of signaling cascades and are involved in a multitude of physiological events important to multicellular organism morphogenesis, ranging from cell adhesion to migration, apoptosis, and angiogenesis as well as pathophysiological behaviors such as those found in cancer metastasis. The transmembrane (TM) domains of integrins are at the center of integrin signaling. Recently, a structure of the TM domains of the aIIbb3 integrin has been reported that sheds light on the signal transduction mechanism of integrins. Integrins are essential TM proteins that couple the extracellular matrix to the cytoskeleton. They consist of noncovalently bound heterodimers, in which each subunit (a and b) contains one TM helix. With eighteen a and eight b subunit types identified, there are 24 known distinct heterodimer combinations with partially overlapping yet specific function. Cells regulate their integrin-mediated adhesion through a variety of mechanisms on different time scales. On the slower time scale, expression patterns are altered by external signals, such as growth factors. On faster time scales, integrins can be redistributed (by clustering or recycling) on the cellular surface, change their intracellular attachment state to the cytoskeleton as exemplified by different lateral mobilities of integrins on a single cell, or alter the affinity state for their extracellular ligand (integrin activation). Changes in the affinity state are correlated with integrin conformational changes, which act as one potential mechanism to relay signals either from the extracellular to the intracellular space (outside-in signaling) or vice versa (inside-out signaling). Integrins are bidirectional signaling molecules, as both signaling directions take place along an allosteric pathway. The two TM helices of the integrin heterodimer are pivotal in signaling events as linkers between the extracellular and intracellular domains. Hence, a variety of groups have pursued different strategies to understand the role of the TM domains in signaling. It was postulated that the TM helices were not merely connectors between the extraand the intracellular space but active structures forming specific heterodimers. However, the structural details of this TM heterodimer have remained elusive until recently. In their recent report, Lau et al. have solved the structure of the aIIbb3 integrin heterodimer in its resting state using highly sophisticated, well-designed NMR spectroscopy experiments on the TM domains in bicelles. This experimentally determined structure allows a solid, structural basis for the prior experimental biochemical results by providing a foundation for an atomistic understanding of TM integrin signaling. The experimental structure shows a right-handed helical dimer (Figure 1). Given that the majority of soluble helix dimers form left-handed structures, this unusual right-

Multiple Approaches Converge on the Structure of the Integrin αIIb/β3 Transmembrane Heterodimer

Integrins link the cytoskeleton to the extracellular matrix and regulate key signaling events that coordinate cellular processes such as secretion, migration, and proliferation. A single integrin molecule can exist in a resting state that does not bind extracellular ligands or in an active state that can engage ligands and form large signaling complexes. Activation signals are transduced between the cytosolic region and the extracellular region by a binary on/off switch in the integrin's transmembrane (TM) domain; the integrin's alpha and beta subunits each have a single TM helix that forms an alpha/beta heterodimer in the resting state, and the TM heterodimer separates to transduce an activation signal across the membrane. In this article, two methods used to generate models of the TM heterodimer, both converging on the same structure, are described. The first model was generated by a Monte Carlo algorithm that selected conformations based on their agreement with published experimental mutagenesis results. The second model was generated by threading the integrin's sequence onto TM helix dimers parsed from the Protein Data Bank and by selecting conformations based on their agreement with published experimental cysteine crosslinking results. The two models have similar structures; however, they differ markedly from some previously published models. To distinguish conformations that reflect the native integrin, we compared the Monte Carlo model, the threaded model, and four published models with experimental mutagenesis and cysteine crosslinking results. The models presented here had high correlation coefficients when compared with experimental findings, and they are in excellent agreement, both in terms of accuracy and in terms of precision, with a recent NMR structure. These results demonstrate that multiple approaches converged on the same structure of the resting integrin's TM heterodimer, and this conformation likely reflects the integrin's native structure.

Self-Assembly of Hydroxy(phenyl)iodonium Ions in Acidic Aqueous Solution: Preparation, and X-ray Crystal Structures of Oligomeric Phenyliodine(III) Sulfates

The treatment of [(diacetoxy)iodo]benzene with sodium bisulfate leads to the formation of oligomeric cationic species resulting from self-assembly of hydroxy(phenyl)iodonium ions, [PhIOH](+), in an aqueous acidic media. Depending on the PhI(OAc)(2):NaHSO(4) ratio, three new oligomeric products have been isolated and characterized by X-ray crystallography. The treatment of 5 equiv of PhI(OAc)(2) with 1 equiv of NaHSO(4).H(2)O affords the previously unknown mu-oxo-[bis(acetoxy)iodo]benzene, PhI(OAc)OI(OAc)Ph, which was structurally characterized by X-ray crystallography. In the solid-state, this mu-oxo product forms an isolated diamond-core dimeric structure with pentagonal-planar iodine centers. The interaction of PhI(OAc)(2) with 1 equiv of NaHSO(4).H(2)O affords a solid-state polymeric phenyliodine(III) sulfate, [(PhIO)(3).SO(3)](n), in which bis(mu-oxo)triiodanyl dication repeat units are linked by sulfate anions. Three of these neighboring polymeric chains form pseudo triple-helix supramolecular structure, which translates along crystallographic b-axis. The reaction of PhI(OAc)(2) with 3 equiv of NaHSO(4).H(2)O results in the formation of a tetrameric phenyliodine(III) sulfate, PhI(OH)OSO(2)O(Ph)IOI(Ph)OSO(2)OI(OH)Ph, the solid state structure of which consists of mu-oxodiiodanyl dicationic species linked by sulfate anions to the terminal hydroxy(phenyl)iodonium groups. The pseudo double-helix structure of this compound formed by the strong intermolecular interactions between two neighbor mu-oxodiiodanyl dicationic fragments and four (per pseudo helix dimer) areas of sulfate anions to the terminal hydroxy(phenyl)iodonium groups interactions.

Utility of surface-supported bilayers in studies of transmembrane helix dimerization

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.

Forces that stabilize membrane proteins

Understanding the energetics of molecular interactions is essential to addressing many of the central quests of biochemistry including protein structure prediction and design, relating structure to function, mapping evolutionary pathways, learning how mutations cause disease and drug design. Hydrogen bonding and Van der Waals packing are two of the fundamental molecular forces that govern the protein structure and function. Because of technical challenges, however, there have been no quantitative tests of these forces in the context of large membrane proteins. While hydrogen-bonding has been widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water, our recent double-mutant cycle analysis shows that the average contribution of eight interhelical side-chain hydrogen-bonding interactions in bacteriorhodopsin is unexpectedly modest, providing only 0.6 kcal of energy per a mol of interaction on an average, which is quite similar to the strengths measured in soluble proteins. Van der Waals packing, on the other hand, is also thought to be important, as highlighted in number of transmembrane helix dimerization motifs that drive strong helix-helix association in the absence of polar residues by providing tight knob-into-hole interactions. Van der Waals strength, reported by the slope of the correlation found between the thermodynamic stability changes that we measured in six individual cavity-creating large-to-small hydrophobic side-chain mutants of bacteriorhodopsin and the increase in cavity size observed in the refined crystal structures of the corresponding mutant proteins, is indeed quite significant and also very similar to the contributions observed in a soluble protein. Weak hydrogen-bonding and significant Van der Waals packing should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.

Folding and Assembly of Membrane Proteins: Coarse Grained Molecular Dynamics Simulations of EmrE

EmrE is a bacterial drug resistance transporter, from E. coli. It is believed to function as an antiparallel homodimer, each monomer of which contains four transmembrane helices. Coarse-grained molecular dynamics (CG-MD) simulations have been previously used to study the insertion and self-assembly of transmembrane helices, and the formation of transmembrane helix dimers and tetramers in lipid bilayers. Such simulations have used a local modification of the original Marrink CG forcefield [1]. In the current study, these methods are employed to investigate the folding and self-assembly of EmrE. Self-assembly CG-MD simulations of the isolated helices of EmrE suggest that each of the constituent helices inserts into a phosphatidylcholine bilayer to adopt a transmembrane orientation. Helix hairpins and other fragments have been simulated to explore the self-assembly and folding processes of the protein subsequent to helix insertion. Simulations of parallel vs. anti-parallel pairs of EmrE monomers are used to explore formation and stability of the EmrE dimer.(1) Bond, P.J., Wee, C.L., and Sansom, M.S.P. (2008) Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer. Biochem. (in press), bi-2008-00642m.R1.

Graph-theoretical identification of dissociation pathways on free energy landscapes of biomolecular interaction

Biomolecular association and dissociation reactions take place on complicated interaction free energy landscapes that are still very hard to characterize computationally. For large enough distances, though, it often suffices to consider the six relative translational and rotational degrees of freedom of the two particles treated as rigid bodies. Here, we computed the six-dimensional free energy surface of a dimer of water-soluble alpha-helices by scanning these six degrees of freedom in about one million grid points. In each point, the relative free energy difference was computed as the sum of the polar and nonpolar solvation free energies of the helix dimer and of the intermolecular coulombic interaction energy. The Dijkstra graph algorithm was then applied to search for the lowest cost dissociation pathways based on a weighted, directed graph, where the vertices represent the grid points, the edges connect the grid points and their neighbors, and the weights are the reaction costs between adjacent pairs of grid points. As an example, the configuration of the bound state was chosen as the source node, and the eight corners of the translational cube were chosen as the destination nodes. With the strong electrostatic interaction of the two helices giving rise to a clearly funnel-shaped energy landscape, the eight lowest-energy cost pathways coming from different orientations converge into a well-defined pathway for association. We believe that the methodology presented here will prove useful for identifying low-energy association and dissociation pathways in future studies of complicated free energy landscapes for biomolecular interaction.

Energetics of Hydrophobic Matching in Lipid-Protein Interactions

Lipid chain length modulates the activity of transmembrane proteins by mismatch between the hydrophobic span of the protein and that of the lipid membrane. Relative binding affinities of lipids with different chain lengths are used to estimate the excess free energy of lipid-protein interaction that arises from hydrophobic mismatch. For a wide range of integral proteins and peptides, the energy cost is much less than the elastic penalty of fully stretching or compressing the lipid chains to achieve complete hydrophobic matching. The chain length dependences of the free energies of lipid association are described by a model that combines elastic chain extension with a free energy term that depends linearly on the extent of residual mismatch. The excess free energy densities involved lie in the region of 0.5–2.0kBT.nm−2. Values of this size could arise from exposure of hydrophobic groups to polar portions of the lipid or protein, but not directly to water, or alternatively from changes in tilt of the transmembrane helices that are energetically comparable to those activating mechanosensitive channels. The influence of hydrophobic mismatch on dimerization of transmembrane helices and their transfer between lipid vesicles, and on shifts in chain-melting transitions of lipid bilayers by incorporated proteins, is analyzed by using the same thermodynamic model. Segmental order parameters confirm that elastic lipid chain distortions are insufficient to compensate fully for the mismatch, but the dependence on chain length with tryptophan-anchored peptides requires that the free energy density of hydrophobic mismatch should increase with increasing extent of mismatch.

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively 15N-labeled peptide containing the TM segment of MCP (MCP TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.

NMR analysis of [ methyl- 13C]methionine UvrB from Bacillus caldotenax reveals UvrB–domain 4 heterodimer formation in solution

UvrB is a central DNA damage recognition protein involved in bacterial nucleotide excision repair. Structural information has been limited by the apparent disorder of the C-terminal domain 4 in crystal structures of intact UvrB; in solution, the isolated domain 4 is found to form a helix–loop–helix dimer. In order to gain insight into the behavior of UvrB in solution, we have performed NMR studies on [ methyl- 13C]methionine-labeled UvrB from Bacillus caldotenax (molecular mass = 75 kDa). The 13 methyl resonances were assigned on the basis of site-directed mutagenesis and domain deletion. Solvent accessibility was assessed based on the relaxation and chemical shift responses of the probe methyl resonances to the stable nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL). M632, located at the potential dimer interface of domain 4, provides an ideal probe for UvrB dimerization behavior. The M632 resonance of UvrB is very broad, consistent with some degree of monomer–dimer exchange and/or conformational instability of the exposed dimer interface. Upon addition of unlabeled domain 4 peptide, the M632 resonance of UvrB sharpens and shifts to a position consistent with a UvrB–domain 4 heterodimer. A dissociation constant ( K D) value of 3.3 μM for the binding constant of UvrB with the domain 4 peptide was derived from surface plasmon resonance studies. Due to the flexibility of the domain 3–4 linker, inferred from limited proteolysis data and from the relaxation behavior of linker residue M607, the position of domain 4 is constrained not by the stiffness of the linking segment but by direct interactions with domains 1–3 in UvrB. In summary, UvrB homodimerization is disfavored, while domain 4 homodimerization and UvrB–domain 4 heterodimerization are allowed.

A mutational study of transmembrane helix–helix interactions

Diverse methods have been developed and applied in the recent years to study interaction of transmembrane α-helices and often interaction of single transmembrane helices is followed on SDS-gels. Here we compare two measurements of the stability of a transmembrane helix–helix interaction, and the stability of the PsbF transmembrane helix dimer was determined in a biological membrane as well as in SDS. The observations described in this study demonstrate that the environment, in which a transmembrane helix interaction is studied, can be very critical and detergent properties can significantly influence transmembrane helix interactions, especially, when the transmembrane domain contains strongly polar residues.