The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH-and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH-dependences determining membrane association and transbilayer insertion transitions are different, but staggered. Anionic lipids not only assist in formation of the insertion competent form, but also lower the kinetic barrier for the final insertion. The function of diphtheria toxin T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome, a task this 178-residue protein is able to perform without the help of any other proteins (1). Although the exact mechanism of membrane translocation is not understood, protein refolding in the lipid bilayer environment has to be the central issue. Thus, deciphering the mechanism of pH-triggered DTT insertion is expected to impact not only the field of cellular entry of toxins, many of which also enter the cell via the *To whom correspondence should be addressed: Phone: 913-588-0489 FAX: 913-588-7440 aladokhin@kumc.edu. † This research was supported by NIH GM-069783 and GM-069783-S1. ‡ Permanent address for Drs. Kyrychenko and Posokhov is Institute for Chemistry at V.N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv, 61077, Ukraine NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript endosomal pathway (2-4), but would also advance our understanding of general physicochemical principles underlying membrane protein assembly and stability.The crystallographic structure of DTT in the water-soluble form (5) (Fig. 1) provides a starting point for refolding/insertion studies. The protein consists of 9 ...

pH-Induced conformational switching is essential for functioning of diphtheria toxin, which undergoes a membrane insertion/translocation transition triggered by endosomal acidification as a key step of cellular entry. In order to establish the sequence of molecular rearrangements and side chain protonation accompanying the formation of the membrane-competent state of the toxin’s translocation (T) domain, we have developed and applied an integrated approach that combines multiple techniques of computational chemistry (e.g., long, µsec-range, all-atom molecular dynamics simulations; continuum electrostatics calculations; and thermodynamic integration) with several experimental techniques of fluorescence spectroscopy. Thermodynamic integration calculations indicate that protonation of H257 causes the greatest destabilization of the native structure (6.9 kcal/mole), which is consistent with our early mutagenesis results. Extensive equilibrium MD simulations with a combined length of over eight µsec demonstrate that histidine protonation, while not accompanied by the loss of structural compactness of the T-domain, nevertheless results in substantial molecular rearrangements characterized by the partial loss of secondary structure due to unfolding of helices TH1 and TH2, and the loss of close contact between the C- and N-terminal segments. The structural changes accompanying the formation of the membrane-competent state ensure an easier exposure of the internal hydrophobic hairpin formed by helices TH8 and TH9, in preparation for its subsequent transmembrane insertion.

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.

Experimental determination of the free energy stabilizing the structure of membrane proteins in their native lipid environment is undermined by the lack of appropriate methods and suitable model systems. Annexin B12 (ANX) is a soluble protein which reversibly inserts into lipid membranes under mildly acidic conditions, which makes it a good experimental model for thermodynamic studies of folding and stability of membrane proteins. Here we apply fluorescence correlation spectroscopy for quantitative analysis of ANX partitioning into large unilamellar vesicles containing either 25% or 75% anionic lipids. Membrane binding of ANX results in changes of autocorrelation time and amplitude, both of which are used in quantitative analysis. The thermodynamic scheme describing acid-induced membrane interactions of ANX considers two independent processes: pH-dependent formation of a membrane-competent form near the membrane interface and its insertion into the lipid bilayer. Our novel fluorescence lifetime topology method demonstrates that the insertion proceeds via an interfacial refolded intermediate state, which can be stabilized by anionic lipids. Lipid titration measurements are used to determine the free energy of both transmembrane insertion and interfacial penetration, which are found to be similar, approximately -10-12 kcal/mol. The formation of the membrane-competent form, examined in a lipid saturation experiment, was found to depend on the local proton concentration near the membrane interface, occurring with pK = 4.3 and involving the protonation of two residues. Our results demonstrate that fluorescence correlation spectroscopy is a convenient tool for the quantitative characterization of the energetics of transmembrane insertion and that pH-triggered ANX insertion is a useful model for studying the thermodynamic stability of membrane proteins.

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of the C-terminal histidines H322, H323 and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by membrane-induced red-shift of tryptophan fluorescence at pH 6.0–6.5. T-domain mutants with replacements at H323 and H372, but not at H322, regain wild type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

The diphtheria toxin T-domain translocates the catalytic C-domain across the endosomal membrane in response to acidification. To elucidate the role of histidine protonation in modulating pH-dependent membrane action of the T-domain, we have used site-directed mutagenesis coupled with spectroscopic and physiological assays. Replacement of H257 with an arginine (but not with a glutamine) resulted in dramatic unfolding of the protein at neutral pH, accompanied by a substantial loss of helical structure and greatly increased exposure of the buried residues W206 and W281. This unfolding and spectral shift could be reversed by the interaction of the H257R mutant with model lipid membranes. Remarkably, this greatly unfolded mutant exhibited WT-like activity in channel formation, N-terminus translocation and cytotoxicity assays. Moreover, membrane permeabilization caused by H257R mutant occurs already at pH 6, where wild type protein is inactive. We conclude that protonation of H257 acts as a major component of the pH-dependent conformational switch, resulting in destabilization of the folded structure in solution and thereby promoting the initial membrane interactions necessary for translocation.

The dimeric OspC/Vsp family surface lipoproteins of Borrelia spirochetes are crucial to the transmission and persistence of Lyme borreliosis and tick-borne relapsing fever. However, the requirements for their proper surface display remained undefined. In previous studies, we showed that localization of Borrelia burgdorferi monomeric surface lipoprotein OspA was dependent on residues in the N-terminal "tether" peptide. Here, site-directed mutagenesis of the B. burgdorferi OspC tether revealed two distinct regions affecting either release from the inner membrane or translocation through the outer membrane. Determinants of both of these steps appear consolidated within a single region of the Borrelia turicatae Vsp1 tether. Periplasmic OspC mutants still were able to form dimers. Their localization defect could be rescued by the addition of an apparently structure-destabilizing C-terminal epitope tag but not by coexpression with wild-type OspC. Furthermore, disruption of intermolecular Vsp1 salt bridges blocked dimerization but not surface localization of the resulting Vsp1 monomers. Together, these results suggest that Borrelia OspC/Vsp1 surface lipoproteins traverse the periplasm and the outer membrane as unfolded monomeric intermediates and assemble into their functional multimeric folds only upon reaching the spirochetal surface.Since the original description of a prokaryotic lipoprotein in the cell envelope of Escherichia coli over 4 decades ago (12), this class of peripherally anchored membrane proteins has been increasingly appreciated. In diderm bacteria, lipoproteins are routed via the general secretory pathway through and to the inner membrane (IM), where they are posttranslationally modified by acylation at a conserved Cys residue (25). Sorting within the periplasm depends on variations of an N-terminal signal first identified in E. coli (23,33,40,62,63,71) and is carried out by the Lol system, consisting of the IM ABC transporter-like LolCDE complex (70), the periplasmic lipoprotein carrier LolA (37), and the outer membrane (OM) lipoprotein receptor LolB (38,72). Established pathways of lipoprotein translocation through the OM involve either a type II or type V secretion system (17,20,51,52,57,69).Beyond the involvement of Braun's lipoprotein Lpp in bacterial cell envelope stability, lipoproteins were shown to play roles in a variety of cellular and pathogenic processes, most recently reviewed in reference 28. In Borrelia spirochetes, the etiologic agents of arthropod-borne Lyme disease and relapsing fever, surface lipoproteins are particularly abundant and constitute the predominant class of known virulence factors at the vector/host-pathogen interface (5,11,29,42,74). Outer surface protein A (OspA), e.g., is expressed by the Lyme disease spirochete Borrelia burgdorferi during the vector phase, where its immunoprotective and adhesive properties appear to ensure continuity of the infectious cycle (6,7,44,45). Upon tick feeding and transmission to a new mammalian host, complex regulatory mechanisms lead t...