Mapping the Structure of an Integral Membrane Protein under Semi-Denaturing Conditions by Laser-Induced Oxidative Labeling and Mass Spectrometry

Little is known about the structural properties of semi-denatured membrane proteins. The current study employs laser-induced oxidative labeling of methionine side chains in combination with electrospray mass spectrometry and optical spectroscopy for gaining insights into the conformation of bacteriorhodopsin (BR) under partially denaturing conditions. The native protein shows extensive oxidation at M32, M68, and M163, which are located in solvent-accessible loops. In contrast, M20 (helix A), M56/60 (helix B), M118 (helix D), M145 (helix E), and M209 (helix G) are strongly protected, consistent with the known protein structure. Exposure of the protein to acidic conditions leads to a labeling pattern very similar to that of the native state. The absence of large-scale conformational changes at low pH is in agreement with recent crystallography data. Solubilization of BR in SDS induces loss of the retinal chromophore concomitant with collapse of the binding pocket, thereby precluding solvent access to the protein interior. Tryptophan fluorescence data confirm the presence of a large protein core that remains protected from water. However, oxidative labeling indicates partial unfolding of helices A and D in SDS. Irreversible thermal denaturation of the protein at 100 °C induces a labeling pattern quite similar to that seen upon SDS exposure. Labeling experiments on refolded bacterioopsin reveal a native-like structure, but with partial unfolding of helix D. Our data suggest that noncovalent contacts with the retinal chromophore in native BR play an important role for the stability of this particular helix. Overall, the present work illustrates the viability of using laser-induced oxidative labeling as a novel tool for characterizing structural changes of membrane proteins in response to alterations of their solvent environment.     

Stability of bacteriorhodopsin alpha-helices and loops analyzed by single-molecule force spectroscopy

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane alpha-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the alpha-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.     

Free energy of membrane protein unfolding derived from single-molecule force measurements

Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.     

Structure of an intermediate in the unfolding of creatine kinase

The homodimeric muscle isoform of creatine kinase (MM-CK) unfolds on exposure to low levels of guanidinium chloride (GdmCl) to yield a partly folded monomeric intermediate. Those regions of MM-CK that experience local unfolding were previously identified through an extensive study of antibody accessibility and protease sensitivity. Since these studies were completed, the coordinates of the rabbit isoform (MM-CK) were released. In light of this, we have determined the minimum changes to this structure required to explain our data on protease and epitope accessibility in the intermediate. We propose that the observed changes occur through (a) disruption of the monomer-monomer interface during dissociation, (b) separation and/ or unfolding of domains or subdomains, and (c) the partial unfolding of solvent-exposed helices. The proposed structure for the intermediate is consistent both with current models of unfolding intermediates and the results of independent studies pertaining to the unfolding of creatine kinase. Proteins 2001;42:269-278.     

Unfolding pathways of native bacteriorhodopsin depend on temperature

The combination of high-resolution atomic force microscopy (AFM) imaging and single-molecule force-spectroscopy was employed to unfold single bacteriorhodopsins (BR) from native purple membrane patches at various physiologically relevant temperatures. The unfolding spectra reveal detailed insight into the stability of individual structural elements of BR against mechanical unfolding. Intermittent states in the unfolding process are associated with the stepwise unfolding of alpha-helices, whereas other states are associated with the unfolding of polypeptide loops connecting the alpha-helices. It was found that the unfolding forces of the secondary structures considerably decreased upon increasing the temperature from 8 to 52 degrees C. Associated with this effect, the probability of individual unfolding pathways of BR was significantly influenced by the temperature. At lower temperatures, transmembrane alpha-helices and extracellular polypeptide loops exhibited sufficient stability to individually establish potential barriers against unfolding, whereas they predominantly unfolded collectively at elevated temperatures. This suggests that increasing the temperature decreases the mechanical stability of secondary structural elements and changes molecular interactions between secondary structures, thereby forcing them to act as grouped structures.     

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu⁹, Glu¹⁹⁴, and Glu²⁰⁴, were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.     

Unfolding pathway of the colicin E1 channel protein on a membrane surface

The channel-forming domain of colicin E1 is composed of a soluble helical bundle which, upon membrane binding, unfolds to form an extended, two-dimensional helical net in the membrane interfacial layer. To characterize the pathway of unfolding of the protein and the structure of the surface-bound intermediate, the time-course of intra-protein distance changes and unfolding on a millisecond time-scale were determined from the kinetics of changes in the efficiency of fluorescence resonance energy transfer, and of the donor-acceptor overlap integral, between each of six individual tryptophan residues and a Cys-conjugated energy transfer acceptor (C509-AEDANS). Comparison of the rate constants revealed the following order of events associated with unfolding of the protein at the membrane surface: (A) movement of the hydrophobic core helices VIII-IX, coincident with a small change in Trp-Cys509 distances of the outer helices; (B) unfolding of surface helices in the helical bundle in the order: helix I, helices III, IV, VI, VII, and helix V; (C) a slow (time-scale, seconds) condensation of the surface-bound helices. The rate of protein unfolding events increased with increasing anionic lipid content. Unfolding did not occur below the lipid thermal phase transition, indicating that unfolding requires mobility in the interfacial layer. The structure of the two-dimensional membrane-bound intermediate in the steady-state was inferred to consist of a quasi-circular arrangement of eight helices embedded in the membrane interfacial layer and anchored by the hydrophobic helical hairpin. The pathway of unfolding of the colicin channel at the membrane surface, catalyzed by electrostatic and hydrophobic forces, is the first described for a membrane-active protein. It is proposed that the pathway and principles described for the colicin protein are relevant to membrane protein import.     

Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.     

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by F?rster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).     

Complex stability of single proteins explored by forced unfolding experiments

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of alpha-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of alpha-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.     

Probing the energy landscape of the membrane protein bacteriorhodopsin

The folding and stability of transmembrane proteins is a fundamental and unsolved biological problem. Here, single bacteriorhodopsin molecules were mechanically unfolded from native purple membranes using atomic force microscopy and force spectroscopy. The energy landscape of individual transmembrane alpha helices and polypeptide loops was mapped by monitoring the pulling speed dependence of the unfolding forces and applying Monte Carlo simulations. Single helices formed independently stable units stabilized by a single potential barrier. Mechanical unfolding of the helices was triggered by 3.9-7.7 A extension, while natural unfolding rates were of the order of 10(-3) s(-1). Besides acting as individually stable units, helices associated pairwise, establishing a collective potential barrier. The unfolding pathways of individual proteins reflect distinct pulling speed-dependent unfolding routes in their energy landscapes. These observations support the two-stage model of membrane protein folding in which alpha helices insert into the membrane as stable units and then assemble into the functional protein.     

Molecular dynamics simulation of the unfolding of individual bacteriorhodopsin helices in sodium dodecyl sulfate micelles

We report molecular dynamics simulations of the trends in the changes in secondary structure of the seven individual helices of bacteriorhodopsin when inserted into sodium dodecyl sulfate (SDS) micelles, and their dependence on the amino acid sequence. The results indicate that the partitioning of the helices in the micelles and their stability are dependent on the hydrophobicity of the transmembrane segments. Helices A, B, and E are stable and retain their initial secondary structure throughout the 100 ns simulation time. In contrast, helices C, D, F, and G show structural perturbations within the first 10 ns. The instabilities are localized near charged residues within the transmembrane segments. The overall structural instability of the helix is correlated with its partitioning to the surface of the micelle and its interaction with polar groups there. The in silico experiments were performed to complement the in vitro experiments that examined the partial denaturation of bacteriorhodopsin in SDS described in the preceding article (DOI 10.1021/bi201769z ). The simulations are consistent with the trends revealed by the experimental results but strongly underestimate the extent of helix to extended coil transformation. The reason may be either that the sampling time was not sufficiently long or, more interestingly, that interhelix residue interactions play a role in the unfolding of the helices.     

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.     

Unfolding of hen egg lysozyme by molecular dynamics simulations at 300K: insight into the role of the interdomain interface

We present the results of two 1.2 ns molecular dynamics (MD) unfolding simulations on hen egg lysozyme in water at 300K, performed using a new procedure called PEDC (Path Exploration With Distance Constraints). This procedure allows exploration of low energy structures as a function of increasing RMSD from the native structure, and offers especially the possibility of extensive exploration of the conformational space during the initial unfolding stages. The two independent MD simulations gave similar chronology of unfolding events: disruption of the active site, kinking of helix C, partial unfolding of the three-stranded beta-sheet to a two-stranded sheet (during which the helices A, B, and D remain to a great extent native), and finally unfolding of the beta-domain and partial unfolding of the alpha-domain in which hydrophobic clusters persist. We show particularly that the loss of hydrophobic contacts between the beta-sheet turn residues Leu55 and Ile56 and the hydrobic patch of the alpha-domain destabilizes the beta-domain and leads to its unfolding, suggesting that the correct embedding of these residues in the alpha-beta interface may constitute the rate limiting step in folding. These results are in accord with experimental observations on the folding/unfolding behavior of hen egg lysozyme at room temperature. They would also explain the loss of stability and the tendency to aggregation observed for the mutant Leu55Thr, and the slow refolding kinetics observed in the analogous amyloidogenic variant of human lysozyme.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

Complex Stability of Single Proteins Explored by Forced Unfolding Experiments

In the last decade atomic force microscopy has been used to measure the mechanical stability of single proteins. These force spectroscopy experiments have shown that many water-soluble and membrane proteins unfold via one or more intermediates. Recently, Li and co-workers found a linear correlation between the unfolding force of the native state and the intermediate in fibronectin, which they suggested indicated the presence of a molecular memory or multiple unfolding pathways (1). Here, we apply two independent methods in combination with Monte Carlo simulations to analyze the unfolding of α-helices E and D of bacteriorhodopsin (BR). We show that correlation analysis of unfolding forces is very sensitive to errors in force calibration of the instrument. In contrast, a comparison of relative forces provides a robust measure for the stability of unfolding intermediates. The proposed approach detects three energetically different states of α-helices E and D in trimeric BR. These states are not observed for monomeric BR and indicate that substantial information is hidden in forced unfolding experiments of single proteins.     

Investigation of the structural stability of hUBF HMG box 5 by native-state hydrogen exchange

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.     

Proton transport by sensory rhodopsins and its modulation by transducer-binding

The study of light-induced proton transfers in the archaeal sensory rhodopsins (SR), phototaxis receptors in Halobacterium salinarum, has contributed important insights into their mechanism of signaling to their cognate transducer subunits in the signaling complex. Essential features of the bacteriorhodopsin (BR) pumping mechanism have been conserved in the evolution of the sensors, which carry out light-driven electrogenic proton transport when their transducers are removed. The interaction of SRI with its transducer blocks proton-conducting channels in the receptor thereby inhibiting its proton pumping, indicating that the pump machinery, rather than the transport activity itself, is functionally important for signaling. Analysis of SRII mutants has shown that the salt bridge between the protonated Schiff base and its counterion Asp73 constrains the receptor in its inactive conformation. Similarly, in BR, the corresponding salt bridge between the protonated Schiff base and Asp85 contributes to constraining the protein in a conformation in which its cytoplasmic channel is closed. Transducer chimera studies further indicate that the receptor conformational changes are transmitted from the sensors to their cognate transducers through transmembrane helix-helix interaction. These and other results reviewed here support a signaling mechanism in which tilting of helices on the cytoplasmic side (primarily outward tilting of helix F), similar to that which occurs in BR in its open cytoplasmic channel conformation, causes structural alterations in the transducer transmembrane helices.     

Identification of Helical Packing Motifs Common to Bacteriorhodopsin and G Protein-Coupled Receptors

A sequence analysis and comparison of transmembrane helices in bacteriorhodopsin (BR) and G protein-coupled receptors (GPCRs) is presented to identify potential regions of homology across protein families. The results show a common pattern of residues is conserved within the interhelical contact regions of BR that fit a knob-into-hole structural motif previously postulated for globular proteins and photosynthetic reaction centers. Based on an alignment of conserved prolines in transmembrane helices, it is inferred that analogous helix packing arrangements are possible in the rhodopsin-like GPCRs. Molecular models of GPCR helices V and VI indicate these interactions occur between aromatic and hydrophobic residues flanking the highly conserved prolines in these sequences. A similar packing arrangement is shown to occur in the X-ray structure of the melittin which also displays a unique pairing of proline-linked helices. The contact pattern identified is further applied to predict the packing of pairs of proline-containing helices in the pheromone-like and cAMP GPCRs. A potential role in stabilizing structure formation is also suggested for the contacts. The results and conclusions are supported by recent biophysical studies of zinc binding to kappa-opioid receptor mutants.     

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.