Associating protein sequence positions with the modulation of quantitative phenotypes.

Although protein sequences encode the information for folding and function, understanding their link is not an easy task. Unluckily, the prediction of how specific amino acids contribute to these features is still considerably impaired. Here, we developed a simple algorithm that finds positions in a protein sequence with potential to modulate the studied quantitative phenotypes. From a few hundred protein sequences, we perform multiple sequence alignments, obtain the per-position pairwise differences for both the sequence and the observed phenotypes, and calculate the correlation between these last two quantities. We tested our methodology with four cases: archaeal Adenylate Kinases and the organisms optimal growth temperatures, microbial rhodopsins and their maximal absorption wavelengths, mammalian myoglobins and their muscular concentration, and inhibition of HIV protease clinical isolates by two different molecules. We found from 3 to 10 positions tightly associated with those phenotypes, depending on the studied case. We showed that these correlations appear using individual positions but an improvement is achieved when the most correlated positions are jointly analyzed. Noteworthy, we performed phenotype predictions using a simple linear model that links per-position divergences and differences in the observed phenotypes. Predictions are comparable to the state-of-art methodologies which, in most of the cases, are far more complex. All of the calculations are obtained at a very low information cost since the only input needed is a multiple sequence alignment of protein sequences with their associated quantitative phenotypes. The diversity of the explored systems makes our work a valuable tool to find sequence determinants of biological activity modulation and to predict various functional features for uncharacterized members of a protein family.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Functional characterization of Legionella pneumophila Cu+ transport ATPase. The activation by Cu+ and ATP.

Cu+-ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu+ transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu+-ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu+-ATPase (LpCopA), the first PIB-ATPase whose structure was determined by X-ray crystallography. Cu+-ATPase activity of the enzyme presents a maximum at ∼37 °C and pH 6.6-6.8. Phospholipids enhance LpCopA Cu+-ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin molar fraction of 0.15 and an amphiphile-protein ratio of ~30,000. As described for other P-ATPases, Mg2+ acts as an essential activator. Furthermore, Cu+-ATPase activity dependence on [Cu+] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu+] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 µM at [Cu+] â†’ ∞) and binding of at least 2 Cu+ with different affinities as well (apparent affinities of 1.4 and 102.5 µM at [ATP] â†’ ∞).

The N-terminal domain of RfaH plays an active role in protein fold-switching.

The bacterial elongation factor RfaH promotes the expression of virulence factors by specifically binding to RNA polymerases (RNAP) paused at a DNA signal. This behavior is unlike that of its paralog NusG, the major representative of the protein family to which RfaH belongs. Both proteins have an N-terminal domain (NTD) bearing an RNAP binding site, yet NusG C-terminal domain (CTD) is folded as a ß-barrel while RfaH CTD is forming an α-hairpin blocking such site. Upon recognition of the specific DNA exposed by RNAP, RfaH is activated via interdomain dissociation and complete CTD structural rearrangement into a ß-barrel structurally identical to NusG CTD. Although RfaH transformation has been extensively characterized computationally, little attention has been given to the role of the NTD in the fold-switching process, as its structure remains unchanged. Here, we used Associative Water-mediated Structure and Energy Model (AWSEM) molecular dynamics to characterize the transformation of RfaH, spotlighting the sequence-dependent effects of NTD on CTD fold stabilization. Umbrella sampling simulations guided by native contacts recapitulate the thermodynamic equilibrium experimentally observed for RfaH and its isolated CTD. Temperature refolding simulations of full-length RfaH show a high success towards α-folded CTD, whereas the NTD interferes with ßCTD folding, becoming trapped in a ß-barrel intermediate. Meanwhile, NusG CTD refolding is unaffected by the presence of RfaH NTD, showing that these NTD-CTD interactions are encoded in RfaH sequence. Altogether, these results suggest that the NTD of RfaH favors the α-folded RfaH by specifically orienting the αCTD upon interdomain binding and by favoring ß-barrel rupture into an intermediate from which fold-switching proceeds.

Conformational dynamics of Tetracenomycin aromatase/cyclase regulate polyketide binding and enzyme aggregation propensity.

BACKGROUND: The N-terminal domain of Tetracenomycin aromatase/cyclase (TcmN), an enzyme derived from Streptomyces glaucescens, is involved in polyketide cyclization, aromatization, and folding. Polyketides are a diverse class of secondary metabolites produced by certain groups of bacteria, fungi, and plants with various pharmaceutical applications. Examples include antibiotics, such as tetracycline, and anticancer drugs, such as doxorubicin. Because TcmN is a promising enzyme for in vitro production of polyketides, it is important to identify conditions that enhance its thermal resistance and optimize its function. METHODS: TcmN unfolding, stability, and dynamics were evaluated by fluorescence spectroscopy, circular dichroism, nuclear magnetic resonance 15N relaxation experiments, and microsecond molecular dynamics (MD) simulations. RESULTS: TcmN thermal resistance was enhanced at low protein and high salt concentrations, was pH-dependent, and denaturation was irreversible. Conformational dynamics on the µs-ms timescale were detected for residues in the substrate-binding cavity, and two predominant conformers representing opened and closed cavity states were observed in the MD simulations. CONCLUSION: Based on the results, a mechanism was proposed in which the thermodynamics and kinetics of the TcmN conformational equilibrium modulate enzyme function by favoring ligand binding and avoiding aggregation. GENERAL SIGNIFICANCE: Understanding the principles underlying TcmN stability and dynamics may help in designing mutants with optimal properties for biotechnological applications.

Folding and Dynamics Are Strongly pH-Dependent in a Psychrophile Frataxin.

Protein dynamics, folding, and thermodynamics represent a central aspect of biophysical chemistry. pH, temperature, and denaturant perturbations inform our understanding of diverse contributors to stability and rates. In this work, we performed a thermodynamic analysis using a combined experimental and computational approach to gain insights into the role of electrostatics in the folding reaction of a psychrophile frataxin variant from Psychromonas ingrahamii. This folding reaction is strongly modulated by pH with a single, narrow, and well-defined transition state with ∼80% compactness, ∼70% electrostatic interactions, and ∼60% hydration shell compared to the native state (αD = 0.82, αH = 0.67, and αΔCp = 0.59). Our results are best explained by a two-proton/two-state model with very different pKa values of the native and denatured states (∼5.5 and ∼8.0, respectively). As a consequence, the stability strongly increases from pH 8.0 to 6.0 (|ΔΔG°| = 5.2 kcal mol-1), mainly because of a decrease in the TΔS°. Variation of ΔH° and ΔS° at pH below 7.0 is dominated by a change in ΔHf⧧ and ΔSf⧧, while at pH above 7.0, it is governed by ΔHu⧧ and ΔSu⧧. Molecular dynamics simulations showed that these pH modulations could be explained by the fluctuations of two regions, rich in electrostatic contacts, whose dynamics are pH-dependent and motions are strongly correlated. Results presented herein contribute to the understanding of the stability and dynamics of this frataxin variant, pointing to an intrinsic feature of the family topology to support different folding mechanisms.

Thermodynamic study of the effect of ions on the interaction between dengue virus NS3 helicase and single stranded RNA.

Dengue virus nonstructural protein 3 (NS3) fulfills multiple essential functions during the viral replication and constitutes a prominent drug target. NS3 is composed by a superfamily-2 RNA helicase domain joined to a serine protease domain. Quantitative fluorescence titrations employing a fluorescein-tagged RNA oligonucleotide were used to investigate the effect of salts on the interaction between NS3 and single stranded RNA (ssRNA). We found a strong dependence of the observed equilibrium binding constant, Kobs, with the salt concentration, decreasing at least 7-fold for a 1-fold increase on cation concentration. As a result of the effective neutralization of ~10 phosphate groups, binding of helicase domain of NS3 to ssRNA is accompanied by the release of 5 or 7 monovalent cations from an oligonucleotide or a polynucleotide, respectively and of 3 divalent cations from the same oligonucleotide. Such estimates are not affected by the type of cation, either monovalent (KCl, NaCl and RbCl) or divalent (MgCl2 and CaCl2), nor by the presence of the protease domain or the fluorescein label. Combined effect of mono and divalent cations was well described by a simple equilibrium binding model which allows to predict the values of Kobs at any concentration of cations.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein.

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

Insights on the conformational dynamics of human frataxin through modifications of loop-1.

Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (µs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the ß-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.

Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin.

Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region.

The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich's Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.

Structural characterization of metal binding to a cold-adapted frataxin.

Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

Chemical changes in bovine serum albumin photoinduced by pterin.

The exposure to UV-A radiation of bovine serum albumin (BSA) in aerated aqueous solution in the presence of pterin (Ptr), results in chemical and conformational modifications of the protein. Ptr belongs to a family of heterocyclic compounds that are well-known type I (electron-transfer) and type II (singlet oxygen) photosensitizers. The evolution of the photosensitized processes was followed by UV/vis spectrophotometry and fluorescence spectroscopy indicating that tryptophan (Trp) and tyrosine (Tyr) residues were affected. Additionally, conformational changes were evaluated by electrophoresis (SDS-PAGE) and size exclusion chromatography coupled with dynamic light scattering detection, showing that BSA undergoes dimerization, via the formation of Tyr radicals. The degradation of Trp residues takes place faster than the oligomerization of the protein. The photosensitized process is initiated by an electron transfer from BSA to the triplet excited stated of Ptr, being a purely dynamic mechanism.

The alteration of the C-terminal region of human frataxin distorts its structural dynamics and function.

Friedreich's ataxia (FRDA) is linked to a deficiency of frataxin (FXN), a mitochondrial protein involved in iron-sulfur cluster synthesis. FXN is a small protein with an α/ß fold followed by the C-terminal region (CTR) with a nonperiodic structure that packs against the protein core. In the present study, we explored the impact of the alteration of the CTR on the stability and dynamics of FXN. We analyzed several pathological and rationally designed CTR mutants using complementary spectroscopic and biophysical approaches. The pathological mutation L198R yields a global destabilization of the structure correlating with a significant and highly localized alteration of dynamics, mainly involving residues that are in contact with L198 in wild-type FXN. Variant FXN 90-195, which is closely related to the FRDA-associated mutant FXN 81-193, conserves a globular shape with a native-like structure. However, the truncation of the CTR results in an extreme alteration of global stability and protein dynamics over a vast range of timescales and encompassing regions far from the CTR, as shown by proton-water exchange rates and (15) N-relaxation measurements. Increased sensitivity to proteolysis, observed in vitro for both mutants, suggests a faster degradation rate in vivo, whereas the enhanced tendency to aggregate exhibited by the truncated variant may account for the loss of functional FXN, with both phenomena providing an explanation as to why the alteration of the CTR causes FRDA. These results contribute to understanding how stability and activity are linked to protein motions and they might be useful for the design of target-specific ligands to control local protein motions for stability enhancement.

Kinetics and thermodynamics of membrane protein folding.

Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane ß-barrel proteins but challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field.

The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin.

The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56-210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81-210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56-81, the moiety putatively responsible for promoting protein-protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or ß-strand structure. In this context, we explored the conformation and stability of hFXN56-210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90-210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81-210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function.

Frataxin from Psychromonas ingrahamii as a model to study stability modulation within the CyaY protein family.

Adaptation of life to low temperatures influences both protein stability and flexibility. Thus, proteins from psychrophilic organisms are excellent models to study relations between these properties. Here we focused on frataxin from Psychromonas ingrahamii (pFXN), an extreme psychrophilic sea ice bacterium that can grow at temperatures as low as -12°C. This α/ß protein is highly conserved and plays a key role in iron homeostasis as an iron chaperone. In contrast to other frataxin homologs, chemical and temperature unfolding experiments showed that the thermodynamic stability of pFXN is strongly modulated by pHs: ranging from 5.5±0.9 (pH6.0) to 0.9±0.3kcalmol(-1) (pH8.0). This protein was crystallized and its X-ray structure solved at 1.45Å. Comparison of B-factor profiles between Escherichia coli and P. ingrahamii frataxin variants (51% of identity) suggests that, although both proteins share the same structural features, their flexibility distribution is different. Molecular dynamics simulations showed that protonation of His44 or His67 in pFXN lowers the mobility of regions encompassing residues 20-30 and the C-terminal end, probably through favorable electrostatic interactions with residues Asp27, Glu42 and Glu99. Since the C-terminal end of the protein is critical for the stabilization of the frataxin fold, the predictions presented may be reporting on the microscopic origin of the decrease in global stability produced near neutral pH in the psychrophilic variant. We propose that suboptimal electrostatic interactions may have been an evolutionary strategy for the adaptation of frataxin flexibility and function to cold environments.

Protein stability and dynamics modulation: the case of human frataxin.

Frataxin (FXN) is an α/ß protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90-195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90-195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90-195 and hFXN90-210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function.

Gain of local structure in an amphipathic peptide does not require a specific tertiary framework.

In this work, we studied how an amphipathic peptide of the surface of the globular protein thioredoxin, TRX94-108, acquires a native-like structure when it becomes involved in an apolar interaction network. We designed peptide variants where the tendency to form alpha-helical conformation is modulated by replacing each of the leucine amino acid residues by an alanine. The induction of structure caused by sodium dodecyl sulfate (SDS) binding was studied by capillary zone electrophoresis, circular dichroism, DOSY-NMR, and molecular dynamics simulations (MDS). In addition, we analyzed the strength of the interaction between a C18 RP-HPLC matrix and the peptides. The results presented here reveal that (a) critical elements in the sequence of the wild-type peptide stabilize a SDS/peptide supramolecular cluster; (b) the hydrophobic nature of the interaction between SDS molecules and the peptide constrains the ensemble of conformations; (c) nonspecific apolar surfaces are sufficient to stabilize peptide secondary structure. Remarkably, MDS shed light on a contact network formed by a limited number of SDS molecules that serves as a structural scaffold preserving the helical conformation of this module. This mechanism might prevail when a peptide with low helical propensity is involved in structure consolidation. We suggest that folding of peptides sharing this feature does not require a preformed tightly-packed protein core. Thus, the formation of specific tertiary interactions would be the consequence of peptide folding and not its cause. In this scenario, folding might be thought of as a process that includes unspecific rounds of structure stabilization guiding the protein to the native state.

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 alpha-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 DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =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.