Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-T_m lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-T_m and low-T_m lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts.     

Regulation of membrane proteins by dietary lipids: effects of cholesterol and docosahexaenoic acid acyl chain-containing phospholipids on rhodopsin stability and function

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (T_m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process (E_a) was determined from DSC thermograms by four separate methods. Both T_m and E_a varied with bilayer composition. Cholesterol increased the T_m both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E_a in the absence of DHA, but raised E_a in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T_m for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability (E_a) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.     

Membrane lateral phase separation induced by proteins of the prothrombinase complex

Blood coagulation factors X and V, as well as prothrombin fragment 1 caused changes in the observed transition temperature (T_m) of appropriately constituted phospholipid vesicles upon binding to the membrane surface. Factor X- and prothrombin fragment 1-induced T_m shifts were calcium-dependent, while factor V changed the T_m in a calcium-independent manner. The results were consistent with clustering of the acidic phospholipid molecules due to protein binding. In all cases, protein binding to acidic phospholipid-containing vesicles caused the observed T_m to approach that for the neutral phospholipid. This resulted in a T_m increase for phospholipid mixtures containing bovine brain phosphatidylserine (PS) plus dipalmitoylphosphatidylcholine (DPPC) and a T_m decrease for mixtures of dipalmitoylphosphatidic acid (DPPA) and dimyristoylphosphatidylcholine (DMPC). Maximum T_m shifts induced in PS-DPPC (10:90) vesicles were very similar for all the prothrombinase proteins and the extent of the change was proportional to the actual amount of membrane-bound protein as determined by light-scattering techniques. For the vitamin K-dependent proteins, T_m changes were greater in the presence of protein plus calcium than in the presence of calcium alone, indicating that lateral phase separation occurs subsequent to initial protein-membrane contact. Lateral phase separation of acidic phospholipids appears to be an important process in the formation of the prothrombinase complex.     

Molecular Dynamics Simulations of a Membrane Protein/Amphipol Complex

Amphipathic polymers known as “amphipols” provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.     

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu⁺ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔG_w° = 12.9 kJ mol^− 1, m = 4.1 kJ mol^− 1 M^− 1, C_m = 3 M, and ΔCp_w° = 0.93 kJ mol^− 1 K^− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.     

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the ¹³C, ¹⁵N, ²H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, H_N, CO, C_α, and C_β chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T _1Z and T₂ relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein.     

Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (K _m and k _cat), thermodynamic stability (T _m and ΔH _m) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.     

Designer Amphiphilic Short Peptides Enhance Thermal Stability of Isolated Photosystem-I

Stability of membrane protein is crucial during protein purification and crystallization as well as in the fabrication of protein-based devices. Several recent studies have examined how various surfactants can stabilize membrane proteins out of their native membrane environment. However, there is still no single surfactant that can be universally employed for all membrane proteins. Because of the lack of knowledge on the interaction between surfactants and membrane proteins, the choice of a surfactant for a specific membrane protein remains purely empirical. Here we report that a group of short amphiphilic peptides improve the thermal stability of the multi-domain protein complex photosystem-I (PS-I) in aqueous solution and that the peptide surfactants have obvious advantages over other commonly used alkyl chain based surfactants. Of all the short peptides studied, Ac-I₅K₂-CONH₂ (I₅K₂) showed the best stabilizing effect by enhancing the melting temperature of PS-I from 48.0°C to 53.0°C at concentration of 0.65 mM and extending the half life of isolated PS-I significantly. AFM experiments showed that PS-I/I₅K₂/Triton X-100 formed large and stable vesicles and thus provide interfacial environment mimicking that of native membranes, which may partly explain why I₅K₂ enhanced the thermal stability of PS-I. Hydrophobic and hydrophilic group length of I_xK_y had an important influence on the stabilization of PS-I. Our results showed that longer hydrophobic group was more effective in stabilizing PS-I. These simple short peptides therefore exhibit significant potential for applications in membrane protein studies.     

Thermodynamic System Drift in Protein Evolution

Proteins from thermophiles are generally more thermostable than their mesophilic homologs, but little is known about the evolutionary process driving these differences. Here we attempt to understand how the diverse thermostabilities of bacterial ribonuclease H1 (RNH) proteins evolved. RNH proteins from Thermus thermophilus (ttRNH) and Escherichia coli (ecRNH) share similar structures but differ in melting temperature (T_m) by 20°C. ttRNH''s greater stability is caused in part by the presence of residual structure in the unfolded state, which results in a low heat capacity of unfolding (ΔC_p) relative to ecRNH. We first characterized RNH proteins from a variety of extant bacteria and found that T_m correlates with the species'' growth temperatures, consistent with environmental selection for stability. We then used ancestral sequence reconstruction to statistically infer evolutionary intermediates along lineages leading to ecRNH and ttRNH from their common ancestor, which existed approximately 3 billion years ago. Finally, we synthesized and experimentally characterized these intermediates. The shared ancestor has a melting temperature between those of ttRNH and ecRNH; the T_ms of intermediate ancestors along the ttRNH lineage increased gradually over time, while the ecRNH lineage exhibited an abrupt drop in T_m followed by relatively little change. To determine whether the underlying mechanisms for thermostability correlate with the changes in T_m, we measured the thermodynamic basis for stabilization—ΔC_p and other thermodynamic parameters—for each of the ancestors. We observed that, while the T_m changes smoothly, the mechanistic basis for stability fluctuates over evolutionary time. Thus, even while overall stability appears to be strongly driven by selection, the proteins explored a wide variety of mechanisms of stabilization, a phenomenon we call “thermodynamic system drift.” This suggests that even on lineages with strong selection to increase stability, proteins have wide latitude to explore sequence space, generating biophysical diversity and potentially opening new evolutionary pathways.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl,urea, and pH

The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near T_m is controlled by kinetics. The values for the thermal transition, T_m, at slow heating scan rate were 66.8, 61.1, and 47.2 °C in the presence of 0.5, 1, and 2 M guanidinium–HCl, respectively. The extrapolated value for T_m in the absence of guanidinium–HCl is 73.7 °C, compared with 70.2 °C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, C_p, at T_m and E_a for the thermal transition decrease under conditions where T_m is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing T_m, but urea reduces C_p relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 °C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.     

Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands

The inherent instability of heptahelical G protein-coupled receptors (GPCRs) during purification and reconstitution is a primary impediment to biophysical studies and to obtaining high-resolution crystal structures. New approaches to stabilizing receptors during purification and screening reconstitution procedures are needed. Here we report the development of a novel homogeneous time-resolved fluorescence assay (HTRF) to quantify properly folded CC-chemokine receptor 5 (CCR5). The assay permits high-throughput thermal stability measurements of femtomole quantities of CCR5 in detergent and in engineered nanoscale apolipoprotein-bound bilayer (NABB) particles. We show that recombinantly expressed CCR5 can be incorporated into NABB particles in high yield, resulting in greater thermal stability compared with that of CCR5 in a detergent solution. We also demonstrate that binding of CCR5 to the HIV-1 cellular entry inhibitors maraviroc, AD101, CMPD 167, and vicriviroc dramatically increases receptor stability. The HTRF assay technology reported here is applicable to other membrane proteins and could greatly facilitate structural studies of GPCRs.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

Coupling of Membrane Nanodomain Formation and Enhanced Electroporation near Phase Transition

Biological cells are enveloped by a heterogeneous lipid bilayer that prevents the uncontrolled exchange of substances between the cell interior and its environment. In particular, membranes act as a continuous barrier for salt and macromolecules to ensure proper physiological functions within the cell. However, it has been shown that membrane permeability strongly depends on temperature and, for phospholipid bilayers, displays a maximum at the transition between the gel and fluid phase. Here, extensive molecular dynamics simulations of dipalmitoylphosphatidylcholine bilayers were employed to characterize the membrane structure and dynamics close to phase transition, as well as its stability with respect to an external electric field. Atomistic simulations revealed the dynamic appearance and disappearance of spatially related nanometer-sized thick ordered and thin interdigitating domains in a fluid-like bilayer close to the phase transition temperature (T_m). These structures likely represent metastable precursors of the ripple phase that vanished at increased temperatures. Similarly, a two-phase bilayer with coexisting gel and fluid domains featured a thickness minimum at the interface because of splaying and interdigitating lipids. For all systems, application of an external electric field revealed a reduced bilayer stability with respect to pore formation for temperatures close to T_m. Pore formation occurred exclusively in thin interdigitating membrane nanodomains. These findings provide a link between the increased membrane permeability and the structural heterogeneity close to phase transition.     

Calcium-dependent Regulation of SNARE-mediated Membrane Fusion by Calmodulin

Neuroexocytosis requires SNARE proteins, which assemble into trans complexes at the synaptic vesicle/plasma membrane interface and mediate bilayer fusion. Ca²⁺ sensitivity is thought to be conferred by synaptotagmin, although the ubiquitous Ca²⁺-effector calmodulin has also been implicated in SNARE-dependent membrane fusion. To examine the molecular mechanisms involved, we examined the direct action of calmodulin and synaptotagmin in vitro, using fluorescence resonance energy transfer to assay lipid mixing between target- and vesicle-SNARE liposomes. Ca²⁺/calmodulin inhibited SNARE assembly and membrane fusion by binding to two distinct motifs located in the membrane-proximal regions of VAMP2 (K_D = 500 nm) and syntaxin 1 (K_D = 2 μm). In contrast, fusion was increased by full-length synaptotagmin 1 anchored in vesicle-SNARE liposomes. When synaptotagmin and calmodulin were combined, synaptotagmin overcame the inhibitory effects of calmodulin. Furthermore, synaptotagmin displaced calmodulin binding to target-SNAREs. These findings suggest that two distinct Ca²⁺ sensors act antagonistically in SNARE-mediated fusion.     

Membrane insertion pathway of annexin B12: thermodynamic and kinetic characterization by fluorescence correlation spectroscopy and fluorescence quenching

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.     

Thermal and pH Stability of the B-Phycoerythrin from the Red Algae Porphyridium cruentum

Phycoerythrin is the major light-harvesting pigment-protein of the red algae Porphyridium cruentum and is widely used as fluorescent probe and analytical reagent. Additionally this protein has a potential application as natural dye in food industry. Nevertheless the knowledge of the functional properties of this alga protein is limited, hindering its application as food additive. In this article we report a biophysical characterization of B-phycoerythrin from Porphyridium cruentum (B-PE) in order to study its stability and spectral properties in a broad range of pHs. This information can help in its potential application as colorant in the food industry. Spectroscopic data obtained in this work show that B-PE has a stronger functional stability in the pH range 4.0–10.0, and Size Exclusion Chromatography suggests that the protein maintains a (αβ)₆-γ oligomeric structure in that range of pHs. At pH 7.0, an apparent T _m value of 77.5?±?0.5 °C was calculated. At this pH, the protein is highly stable with a loss of only 20 % of its spectral properties (absorbance and fluorescence) after 25 days at room temperature. These results indicate that B-PE is more stable in a broad range of pHs than other phycoerythrin proteins, which would facilitate its use in the food industry.     

Membrane proteins enter the fold

Membrane proteins have historically been recalcitrant to biophysical folding studies. However, recent adaptations of methods from the soluble protein folding field have found success in their applications to transmembrane proteins composed of both α-helical and β-barrel conformations. Avoiding aggregation is critical for the success of these experiments. Altogether these studies are leading to discoveries of folding trajectories, foundational stabilizing forces and better-defined endpoints that enable more accurate interpretation of thermodynamic data. Increased information on membrane protein folding in the cell shows that the emerging biophysical principles are largely recapitulated even in the complex biological environment.     

Membrane phase behavior of Escherichia coli during desiccation, rehydration, and growth recovery

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T_m), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T_m from 8.4 ± 1.7 °C (in undried control samples) to 16.5 ± 1.3 °C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T_m, which began approximately 1 day after initial rehydration and plateaued at 12.5 ± 1.8 °C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T_m and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.