8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.     

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in sign ificantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.     

Identification of amino acids involved in protein structural uniqueness: implication for de novo protein design

Structural uniqueness is characteristic of native proteins and is essential to express their biological functions. The major factors that bring about the uniqueness are specific interactions between hydrophobic residues and their unique packing in the protein core. To find the origin of the uniqueness in their amino acid sequences, we analyzed the distribution of the side chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived deltaS(contact), the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure. The deltaS(contact) values indicate distinct tendencies of the residue pairs to restrict side chain conformation by inter-residue contacts. Of the hydrophobic residues in alpha-helices, aliphatic residues (Leu, Val, Ile) strongly restrict the side chain conformations of each other. In beta-sheets, Met is most strongly restricted by contact with Ile, whereas Leu, Val and Ile are less affected by other residues in contact than those in alpha-helices. In designed and native protein variants, deltaS(contact) was found to correlate with the folding-unfolding cooperativity. Thus, it can be used as a specificity parameter for designing artificial proteins with a unique structure.     

Identification of the dimerization domain of dehalogenase IVa of Burkholderia cepacia MBA4

Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.     

Purification and characterization of Rhodobacter sphaeroides acyl carrier protein

Acyl carrier protein (ACP) has been purified from the facultative phototrophic bacterium Rhodobacter sphaeroides. The ACP preparation was greater than 95% homogeneous as determined by native and disodium dodecyl sulfate (Na2DodSO4)-polyacrylamide gel electrophoreses and N-terminal amino acid analysis. Amino acid compositional analysis revealed that the protein contains approximately 75 amino acids, has a calculated minimum molecular weight of 8700, and lacks the amino acids tyrosine and tryptophan. The presence of the characteristic 4'-phosphopantetheine prosthetic group was indicated by the occurrence of equimolar quantities of beta-alanine and taurine in amino acid hydrolysates and was confirmed by independent chemical analysis. The protein displayed a pI of 3.8 and had a calculated partial specific volume of 0.732 mL/g. The primary structure of the protein has been determined for the first 46 amino acid residues from the N terminus of the molecule, and the region of the molecule encompassing the amino acids from residues 31 to 44 was found to have 100% homology with the identical residues in Escherichia coli ACP. In contrast to E. coli ACP, R. sphaeroides ACP migrated according to its molecular weight during Na2DodSO4 gel electrophoresis, was resistant to pH-induced denaturation, and comigrated with the cis-vaccenoyl-ACP derivative during native gel electrophoresis. It is proposed that the basis for these properties is the enhanced hydrophobic character of the protein.     

Local propensities and statistical potentials of backbone dihedral angles in proteins

The following three issues concerning the backbone dihedral angles of protein structures are presented. (1) How do the dihedral angles of the 20 amino acids depend on the identity and conformation of their nearest residues? (2) To what extent are the native dihedral angles determined by local (dihedral) potentials? (3) How to build a knowledge-based potential for a residue's dihedral angles, considering the identity and conformation of its nearest residues? We find that the dihedral angle distribution for a residue can significantly depend on the identity and conformation of its adjacent residues. These correlations are in sharp contrast to the Flory isolated-pair hypothesis. Statistical potentials are built for all combinations of residue triplets and depend on the dihedral angles between consecutive residues. First, a low-resolution potential is obtained, which only differentiates between the main populated basins in the dihedral angle density plots. Minimization of the dihedral potential for 125 test proteins reveals that most native alpha-helical residues (89%) and a large fraction of native beta-sheet residues (47%) adopt conformations close to their native one. For native loop residues, the percentage is 48%. It is also found that this fraction is higher for residues away from the ends of alpha or beta secondary structure elements. In addition, a higher resolution potential is built as a function of dihedral angles by a smoothing procedure and continuous functions interpolations. Monte Carlo energy minimization with this potential results in a lower fraction for native beta-sheet residues. Nevertheless, because of the higher flexibility and entropy of beta structures, they could be preferred under the influence of non-local interactions. In general, most alpha-helices and many beta-sheets are strongly determined by the local potential, while the conformations in loops and near the end of beta-sheets are more influenced by non-local interactions.     

High polar content of long buried blocks of sequence in protein domains suggests selection against amyloidogenic non-polar sequences

Native protein structures achieve stability in part by burying hydrophobic side-chains. About 75% of all amino acid residues buried in protein interiors are non-polar. Buried residues are not uniformly distributed in protein sequences, but sometimes cluster as contiguous polypeptide stretches that run through the interior of protein domain structures. Such regions have an intrinsically high local sequence density of non-polar residues, creating a potential problem: local non-polar sequences also promote protein misfolding and aggregation into non-native structures such as the amyloid fibrils in Alzheimer's disease. Here we show that long buried blocks of sequence in protein domains of known structure have, on average, a lower content of non-polar amino acids (about 70%) than do isolated buried residues (about 80%). This trend is observed both in small and in large protein domains and is independent of secondary structure. Long, completely non-polar buried stretches containing many large side-chains are particularly avoided. Aspartate residues that are incorporated in long buried stretches were found to make fewer polar interactions than those in short stretches, hinting that they may be destabilizing to the native state. We suggest that evolutionary pressure is acting on non-native properties, causing buried polar residues to be placed at positions where they would break up aggregation-prone non-polar sequences, perhaps even at some cost to native state stability.     

Cloning,expression, and assembly of sericin-like protein

Recombinant sericin proteins of different molecular masses (17.4, 31.9, and 46.5 kDa), based on the 38-amino acid repetitive motif of native sericin, were cloned, expressed, and purified. The recombinant sericin self-assembled during dialysis (starting concentration of 2.5 mg/ml) forming twisted fibers. Circular dichroism and Fourier transform infrared spectroscopy studies demonstrated protein conformational transitions occurred from random coil to beta-sheets during the dialysis. Congo red-stained recombinant sericin fibrils exhibited apple-green birefringence, indicating long-range order in the array of beta-sheets. Biosynthetic sericin has a high content of polar amino acids (e.g. > 40 mol % serine), leading to a beta-sheet conformation formed by hydrogen bonding via polar zipper interactions. Analysis of recombinant sericin sequence using Mandel-Gutfreund's (Mandel-Gutfreund, Y., and Gregoret, L. M. (2002) J. Mol. Biol. 323, 453-461) definition of polar and non-polar amino acids showed that the hydrophobicity pattern resembles the most frequent pattern of amyloidogenic proteins, polar amino acid aggregates (PPPPP). Many beta-proteins and peptides are designed to study amyloidogenesis using a polar/non-polar alternating pattern (PNPNPN). Sericin-like proteins or peptides provide an alternative model in terms of hydrophobicity pattern with which to explore questions related to beta-sheet formation and amyloidogenesis. The glue-like property of sericin is attributed to the hydrogen bonding between serine residues of sericin with serine residues in the fibroin structural components of silk fiber.     

Characterization of a partially folded intermediate of stem bromelain at low pH.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions.     

Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.     

NMR characterizations of an amyloidogenic conformational ensemble of the PI3K SH3 domain

Amyloid formation is associated with structural changes of native polypeptides to monomeric intermediate states and their self-assembly into insoluble aggregates. Characterizations of the amyloidogenic intermediate state are, therefore, of great importance in understanding the early stage of amyloidogenesis. Here, we present NMR investigations of the structural and dynamic properties of the acid-unfolded amyloidogenic intermediate state of the phosphatidylinositol 3-kinase (PI3K) SH3 domain--a model peptide. The monomeric amyloidogenic state of the SH3 domain studied at pH 2.0 (35 degrees C) was shown to be substantially disordered with no secondary structural preferences. (15)N NMR relaxation experiments indicated that the unfolded polypeptide is highly flexible on a subnanosecond timescale when observed under the amyloidogenic condition (pH 2.0, 35 degrees C). However, more restricted motions were detected in residues located primarily in the beta-strands as well as in a loop in the native fold. In addition, nonnative long-range interactions were observed between the residues with the reduced flexibility by paramagnetic relaxation enhancement (PRE) experiments. These indicate that the acid-unfolded state of the SH3 domain adopts a partly folded conformation through nonnative long-range contacts between the dynamically restricted residues at the amyloid-forming condition.     

Positive and negative design in stability and thermal adaptation of natural proteins

The aim of this work is to elucidate how physical principles of protein design are reflected in natural sequences that evolved in response to the thermal conditions of the environment. Using an exactly solvable lattice model, we design sequences with selected thermal properties. Compositional analysis of designed model sequences and natural proteomes reveals a specific trend in amino acid compositions in response to the requirement of stability at elevated environmental temperature: the increase of fractions of hydrophobic and charged amino acid residues at the expense of polar ones. We show that this “from both ends of the hydrophobicity scale” trend is due to positive (to stabilize the native state) and negative (to destabilize misfolded states) components of protein design. Negative design strengthens specific repulsive non-native interactions that appear in misfolded structures. A pressure to preserve specific repulsive interactions in non-native conformations may result in correlated mutations between amino acids that are far apart in the native state but may be in contact in misfolded conformations. Such correlated mutations are indeed found in TIM barrel and other proteins.     

On the DNA binding protein II from Bacillus stearothermophilus. II. The amino acid sequence and its relation to those of homologous proteins from other prokaryotes

The complete amino acid sequence of DNA binding protein II from Bacillus stearothermophilus has been determined. The protein contains 90 amino acid residues and has a calculated Mr of 9716. The sequence is compared to homologous molecules from Escherichia coli, Thermoplasma acidophilum, and Pseudomonas aeruginosa (where only a partial sequence is available). The B. stearothermophilus molecule has 58% and 59% residues identical with the two forms of the E. coli protein and 32% with the T. acidophilum protein. There are totally conserved residues at positions 46-48 and 61-65 with an intervening cluster of basic amino acids in all four proteins.     

Dynamics in the unfolded state of beta2-microglobulin studied by NMR

Many proteins form amyloid-like fibrils in vitro under conditions that favour the population of partially folded conformations or denatured state ensembles. Characterising the structural and dynamic properties of these states is crucial towards understanding the mechanisms of self-assembly in amyloidosis. The aggregation of beta2-microglobulin (beta2m) into amyloid fibrils in vivo occurs in the condition known as dialysis-related amyloidosis (DRA) and the protein has been shown to form amyloid-like fibrils under acidic conditions in vitro. We have used a number of 1H-15N nuclear magnetic resonance (NMR) experiments in conjunction with site-directed mutagenesis to study the acid-unfolded state of beta2m. 15N NMR transverse relaxation experiments reveal that the acid-denatured ensemble, although predominantly unfolded at the N and C termini, contains substantial non-native structure in the central region of the polypeptide chain, stabilised by long-range interactions between aromatic residues and by the single disulphide bond. Relaxation dispersion studies indicate that the acid-unfolded ensemble involves two or more distinct species in conformational equilibrium on the micro- to millisecond time-scale. One of these species appears to be hydrophobically collapsed, as mutations in an aromatic-rich region of the protein, including residues that are solvent-exposed in the native protein, disrupt this structure and cause a consequent decrease in the population of this conformer. Thus, acid-unfolded beta2m consists of a heterogeneous ensemble of rapidly fluctuating species, some of which contain stable, non-native hydrophobic clusters. Given that amyloid assembly of beta2m proceeds with lag kinetics under the conditions of this study, a rarely populated species such as a conformer with non-native aromatic clustering could be key to the initiation of amyloidosis.     

Beta-sheet capping: signals that initiate and terminate beta-sheet formation

In the present work, we address the question of whether different amino acids have different beta-sheet initiating and terminating characteristics. Using a large scale analysis of parallel and antiparallel beta-sheets in a non-redundant dataset of proteins, we observed that most of the amino acids show significant under- or over-representation in at least one of the positions at the two ends of beta-sheets, which are denoted as N-cap and C-cap. In addition, based on statistical data and structural comparison, we found that certain amino acids, especially Asp, Asn, Gly and Pro have strong tendencies to block beta-sheet continuation. Hence, we can consider these residues as beta-sheet terminators. It was also proposed that the dipole moments in parallel beta-sheets, whose direction is from C-terminal (partially negative) to N-terminal (partially positive), are much stronger than has previously been suggested. In fact, enhancement of dipole moments in parallel beta-sheets is a result of the positioning of positively charged residues at N-cap and negatively charged residues at C-cap. This enhancement in dipole moment magnitude leads to strengthened dipolar interactions between parallel beta-sheets dipoles and other partners especially alpha-helices dipoles. The results provide an explanation for the antiparallel alignment of parallel beta-sheets with alpha-helices.     

Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp. GL1 at 1.8 A resolution

Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).     

Crystal structure analysis of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis

The structure of acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis has been determined by X-ray diffraction analysis using MAD data in a crystal of space group P2?, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) ?, β = 99.49 (3) ° and been refined to a crystallographic R-factor of 17.4% and R-free of 23.7%. It contains two dimers in one asymmetric unit, in which native Amds/Fmds (TE19) contains of the 32 kDa native protein. The final model consists of 4 monomer (299 amino acids residues with additional 2 expression tag amino acids residues), 5 Ca2?, 4 Zn2? and 853 water molecules. The monomer is composed by the following: an N-domain which is featuring by three-layers β/β/β; a prominent excursion between N-terminal end of strand β? and β??, which contains four-stranded antiparallel β sheet; an C-domain which is formed by the last 82 amino acid residues with the feature of mixed α/β structure. The protein contains ion-pair Ca2?-Zn2?. The portion of three-layer β/β/β along with the loops provides four protein ligands to the tightly bound Ca2?, three water molecules complete the coordination; and provides five protein ligands to the tightly bound Zn2?, one water molecule complete the coordination.     

Anion-induced folding of rabbit muscle pyruvate kinase: existence of multiple intermediate conformations at low pH

Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated under neutral as well as acidic conditions by using enzymatic activity measurements and a combination of optical methods, such as circular dichroism, fluorescence, and ANS binding. At low pH and low ionic strength, pyruvate kinase exists in a partially unfolded state (UA state) retaining half of the secondary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na2SO4, to the acid-unfolded state induces refolding, resulting structural propensities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and decrease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to the intermolecular association. The anion-refolded state is more stable than the UA state, and its stability is nearly equal to that of native protein toward chemical-induced unfolding by Gu-HCl and urea. Moreover, at low concentrations, Gu-HCl behaves like an anion, by inducing refolding of the acid-unfolded state with structural features equivalent to that of native molecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability.     

Crystal structure of rat intestinal fatty-acid-binding protein. Refinement and analysis of the Escherichia coli-derived protein with bound palmitate

Rat intestinal fatty-acid-binding protein (I-FABP) is a small (15,124 Mr) cytoplasmic polypeptide that binds long-chain fatty acids in a non-covalent fashion. I-FABP is a member of a family of intracellular binding proteins that are thought to participate in the uptake, transport and/or metabolic targeting of hydrophobic ligands. The crystal structure of Escherichia coli-derived rat I-FABP with a single molecule of bound palmitate has been refined to 2 A resolution using a combination of least-squares methods, energy refinement and molecular dynamics. The combined methods resulted in a model with a crystallographic R-factor of 17.8% (7775 reflections, sigma greater than 2.0), root-mean-square bond length deviation of 0.009 A and root-mean-square bond angle deviation of 2.85 degrees. I-FABP contains ten antiparallel beta-strands organized into two approximately orthogonal, beta-sheets. The hydrocarbon tail of its single C16:0 ligand is present in a well-ordered, distinctively bent conformation. The carboxylate group of the fatty acid is located in the interior of I-FABP and forms a unique "quintet" of electrostatic interactions involving Arg106; Gln 115, and two solvent molecules. The hydrocarbon tail is bent with a slight left-handed helical twist from the carboxylate group to C-16. The bent methylene chain resides in a "cradle" formed by the side-chains of hydrophobic, mainly aromatic, amino acid residues. The refined molecular model of holo-I-FABP suggests several potential locations for entry and exiting of the fatty acid.     

Equilibrium titrations of acid-induced unfolding-refolding and salt-induced molten globule of cytochrome c by FT-IR spectroscopy

Despite extensive investigations on the acid-unfolded and acid/salt-induced molten globule(-like) states of cytochrome c using variety of techniques, structural features of the acid-unfolded state in terms of residual secondary structures and the structural transition between the acid-unfolded and acid/salt-refolded states have not been fully characterized beyond the circular dichroism (CD) spectroscopy. It is unusual that secondary structure(s) of the unfolded state leading to the molten globule state, an important protein folding intermediate, as determined by CD was not fully corroborated by independent experimental method(s). In this study, we carried out an equilibrium titration of acid-induced unfolding and subsequent acid- and salt-induced refolding of cytochrome c using Fourier transform infrared spectroscopy. The spectral profiles of the equilibrium titration reveal new structural details about the acid-unfolded state and the structural transition associated with the acid/salt-refolded molten globule(-like) states of cytochrome c.