Reversible structural changes in a hydrophobic protein,elastin, as indicated by fluorescence probe analysis

Fluorescent probe analysis of purified elastin using 1-anilinonaphthalene-8-sulfonate has been used to investigate reversible structural changes that accompany stretching of this rubberlike protein. There is a specific binding of 1-anilinonaphthalene-8-sulfonate to elastin with a single dye molecule attached per 74,000 molecular-weight protein subunit. When labeled elastin is stretched, the intensity of the 1-anilinonaphthalene-8-sulfonate fluorescence decreases reversibly, and this decrease appears to be linked to an increase in the environmental polarity in the immediate vicinity of the bound dye molecule. The results of experiments carried out in H₂O and D₂O indicate that this polarity change is due to an increase in the exposure of the 1-anilinonaphthalene-8-sulfonate to water as the hydrophobic interior of the protein subunit is unfolded during stretching. The data are consistent with the proposal that the elastin network is a two-phase system of hydrophobic protein globules surrounded by free solvent spaces.     

A fluorimetric study of Mg2]nduced structural changes in thylakoid membrane protein.

Low concentrations of Mg²⁺ (concn < 10 mm) generate structural changes in delipidated spinach chloroplast lamellae, that appear as changes in the fluorescence yield of native tryptophyl residues and of the externally added polarity probe magnesium 1-anilinonaphthalene-8-sulfonate.The delipidated lamellae, consisting essentially of structural protein monomers and aggregates, bind magnesium 1-anilinonaphthalene-8-sulfonate to the extent of 126 ± 13 nmol/mg protein, and with a dissociation constant K_D = 167 μM. Bound ANS fluoresces at 458 nm with a quantum yield Φ = 0.121. Tryptophyls sensitize the fluorescence of bound ANS with a maximal efficiency T_max = 0.85. Assuming completely random orientation of the interacting chromophores, an interchromophore separation $\text{R = 17.3}\text{A}\text{?}$ is calculated. Only two-thirds of the membrane tryptophyls have ANS-binding sites in their vicinity.Mg²⁺ binds to the delipidated membranes with a dissociation constant K_D = 2 mM. The binding is attended by enhancement of magnesium 1-anilinonaphthalene-8-sulfonate fluorescence, and deenhancement of tryptophyl fluorescence, while the efficiency of interchromophore excitation transfer increases only slightly. These effects suggest that Mg²⁺ generates a structural change which lowers the polarity of the membrane region where tryptophyl and magnesium 1-anilinonaphthalene-8-sulfonate are situated, but which has a minor effect only on the interchromophore separation.     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

Interaction of alpha-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution.

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.     

Mildly acidic pH activates the extracellular molecular chaperone clusterin

Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.     

Fluorescence and FTIR study of the pressure-induced denaturation of bovine pancreas trypsin.

The pressure denaturation of trypsin from bovine pancreas was investigated by fluorescence spectroscopy in the pressure range 0. 1-700 MPa and by FTIR spectroscopy up to 1000 MPa. The tryptophan fluorescence measurements indicated that at pH 3.0 and 0 degrees C the pressure denaturation of trypsin is reversible but with a large hysteresis in the renaturation profile. The standard volume changes upon denaturation and renaturation are -78 mL.mol-1 and +73 mL.mol-1, respectively. However, the free energy calculated from the data in the compression and decompression directions are quite different in absolute values with + 36.6 kJ.mol-1 for the denaturation and -5 kJ. mol-1 for the renaturation. For the pressure denaturation at pH 7.3 the tryptophan fluorescence measurement and enzymatic activity assays indicated that the pressure denaturation of trypsin is irreversible. Interestingly, the study on 8-anilinonaphthalene-1-sulfonate (ANS) binding to trypsin under pressure leads to the opposite conclusion that the denaturation is reversible. FTIR spectroscopy was used to follow the changes in secondary structure. The pressure stability data found by fluorescence measurements are confirmed but the denaturation was irreversible at low and high pH in the FTIR investigation. These findings confirm that the trypsin molecule has two domains: one is related to the enzyme active site and the tryptophan residues; the other is related to the ANS binding. This is in agreement with the study on urea unfolding of trypsin and the knowledge of the molecular structure of trypsin.     

A conformational change in bovine beta-lactoglobulin at low pH.

A conformational change at low pH in bovine beta-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate. Both studies show that when the pH of beta-lactoglobulin solutions is altered between 6.5 and 2.0, a rapid change in protein conformation occurs, followed by a slower conformational change. It seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0. The slow changes involve shifts in protein conformation of the region that includes one of the protein tryptophan residues.     

Identification of Acidic pH-dependent Ligands of Pentameric C-reactive Protein

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pH-treated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.     

Changes in the lipid and protein components of thymus cell membrane during lipid peroxidation]

Nonenzymatic lipid peroxidation in thymus cell plasma membranes was studied. The composition of lipid and protein components, intensity of fluorescence of the membrane probes (1-anilinonaphthalene-8-sulfonate, 4-dimethylaminochalcon, eosin, pyronin and rhodamine), fluorescence polarization of tryptophan residues of membrane proteins and quenching by acrylamide of intrinsic fluorescence of proteins were determined. Induction of lipid peroxidation by the Fe(2+)-ascorbate system caused changes in the composition and structure of lipids. This was paralleled with changes in the structural-dynamic organization of membrane proteins, transition of some peripheral proteins to the water phase and increased solubilization of integral proteins by Triton X-100.     

Interstrand loops CD and EF act as pH-dependent gates to regulate fatty acid ligand binding in tear lipocalin

Tear lipocalin (TL), a major component of human tears, shows pH-dependent endogenous ligand binding. The structural and conformational changes associated with ligand release in the pH range of 7.5-3.0 are monitored by circular dichroism spectroscopy and site-directed tryptophan fluorescence. In the transition from pH 7.5 to pH 5.5, the ligand affinity for 16-(9-anthroyloxy)palmitic acid (16AP) and 8-anilino-1-naphthalenesulfonic acid is reduced. At pH 4.0 these ligands no longer bind within the TL calyx. From pH 7.3 to pH 3.0, the residues on loops CD and EF, which overhang the calyx entrance, show reduced accessibility to acrylamide. In addition resonance energy transfer is enhanced between residues on the two loops; the distance between the loops narrows. These findings suggest that apposition of the loops at low pH excludes the ligand from the intracavitary binding site. The conformational changes observed in transition from pH 7.3 to pH 3.0 for loops CD and EF are quite different. The CD loop shows less population reshuffling than the EF loop with an acidic environment, probably because backbone motion is restrained by the adjacent disulfide bond. The Trp fluorescence wavelength maximum (lambda(max)) reflects internal electrostatic interactions for positions on loops CD and EF. The titration curves of lambda(max) for mutants on the EF loop fit the Hendersen-Hasselbalch equation for two apparent pK(a) values, while the CD loop positions fit satisfactorily with one pK(a) value. Midpoints of transition for the binding affinity of TL tryptophan mutants to 16AP occur at pH 5.5-6.1. Replacement of each amino acid on either loop by single tryptophan mutation does not disrupt the pH-dependent binding affinity to 16AP. Taken together the data suggest that pH-driven ligand release involves ionization changes in several titratable residues associated with CD and EF loop apposition and occlusion of the calyx.     

Comparison of the pH-induced conformational change of different clostridial neurotoxins

Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane.     

Photoacoustic analysis of proteins: volumetric signals and fluorescence quantum yields.

A series of proteins has been examined using time-resolved, pulsed-laser volumetric photoacoustic spectroscopy. Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence quantum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples. The proteins studied were the green fluorescent protein (GFP); intestinal fatty acid binding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of this group of proteins, only the ALBP labeled with 1,8-ANS showed significant nonthermal volume changes at the beta = 0 temperature (approximately 3.8 degrees C) for the buffer used (10 mM Tris-HCI, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient). For all of the proteins except for acrylodan-labeled IFABP, the fluorescence quantum yields calculated assuming simple energy conservation were anomalously high, i.e., the apparent heat signals were lower than those predicted from independent fluorescence measurements. The consistent anomalies suggest that the low photoacoustic signals may be characteristic of fluorophores buried in proteins, and that photoacoustic signals derive in part from the microenvironment of the absorbing chromophore.     

Increased flexibility and liposome-binding capacity of CD1e at endosomal pH

The plasma membrane proteins CD1a, CD1b and CD1c are expressed by human dendritic cells, the professional antigen-presenting cells of the immune system, and present lipid antigens to T lymphocytes. CD1e belongs to the same family of molecules, but accumulates as a membrane-associated form in the Golgi compartments of immature dendritic cells and as a soluble cleaved form in the lysosomes of mature dendritic cells. In lysosomes, the N-terminal propeptide of CD1e is also cleaved, but the functional consequences of this step are unknown. Here, we investigated how the pH changes encountered during transport to lysosomes affect the structure of CD1e and its ligand-binding properties. Circular dichroism studies demonstrated that the secondary and tertiary structures of recombinant CD1e were barely altered by pH changes. Nevertheless, at acidic pH, guanidium chloride-induced unfolding of CD1e molecules required lower concentrations of denaturing agent. The nonfunctional L194P allelic variant was found to be structurally less stable at acidic pH than the functional forms, providing an explanation for the lack of its detection in lysosomes. The number of water-exposed hydrophobic patches that bind 8-anilinonaphthalene-1-sulfonate was higher in acidic conditions, especially for the L194P variant. CD1e molecules interacted with lipid surfaces enriched in anionic lipids, such as bis(monoacylglycero)phosphate, a late endosomal/lysosomal lipid, especially at acidic pH, or when the propeptide was present. Altogether, these data indicate that, in the late endosomes/lysosomes of DCs, the acid pH promotes the binding of lipid antigens to CD1e through increased hydrophobic and ionic interactions.     

Partly folded states of bovine carbonic anhydrase interact with zwitterionic and anionic lipid membranes

The acidic, partly folded states of bovine carbonic anhydrase II (BCAII) were used as an experimental system to study the interactions of partly denatured proteins with lipid membranes. The pH dependence of their interactions with palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) membranes was studied. A filtration binding assay shows that acidic partly folded states of BCAII bind to POPC membranes. Fluorescence emission spectra from Trp residues of the bound protein are slightly shifted to shorter wavelength and can be quenched by a water-soluble quencher of fluorescence, indicating that the binding occurs without deep penetration of Trp residues into the membrane. The content of beta-structures of the protein in solution, as revealed by FT-IR spectroscopy, decreases in the partly folded states and the binding to POPC membrane occurs without further changes of secondary structure. In the presence of 0.1 M NaCl, a partly folded state self-aggregates and does not bind to POPC membrane. At acidic pH, BCAII binds to POPG membranes both at high and low ionic strength. The binding to the anionic lipid occurs with protein self-aggregation within the lipid-protein complexes and with changes in the secondary structure; large blue shifts in the fluorescence emission spectra and the decrease in the exposure to water-soluble acrylamide quencher of Trp fluorescence strongly suggest that BCAII penetrates the hydrocarbon domain in the POPG-protein complexes.     

A reassessment of copper(II) binding in the full-length prion protein

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.     

Folding Study of Venus Reveals a Strong Ion Dependence of Its Yellow Fluorescence under Mildly Acidic Conditions

Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of ¹⁵N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the β-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.     

Calcium-induced conformational changes in Leishmania infantum kinetoplastid membrane protein-11

Total-reflection X-ray fluorescence has been used to study whether the Leishmania infantum kinetoplastid membrane protein-11 is a Ca2+-binding protein. The 108 amino acid helix-loop-helix protein has the loop region located between residues 45 and 57, having similarity to the EF-hand motifs. In particular, the sequence alignment of the putative motif revealed the existence of 67% similarity and 33% identity with the EF-hand of the plasmodia-specific 40-kDa protein from Physarum polycephalum. To address the type of conformational changes induced by Ca2+ binding, circular dichroism and fluorescence spectroscopy were used. The data showed that Ca2+ induces changes in both the secondary and tertiary structure of the protein in a temperature- and pH-dependent way. It also induces the precipitation of the protein at pH 7.5, in contrast with what occurs at pH 5.0, and the precipitation process can be reverted by addition of EGTA. At acidic pH values the complex EGTA-Ca2+ causes drastic structural changes, forcing the protein to adopt a structure close to that of a random coil. Because, at acidic pH values, protein:Ca2+:EGTA ternary complexes may be formed, the drastic change may be attributed to the presence of a high density of EGTA negative charges in the neighborhood of the alpha-helices.     

Dissociation and unfolding of bovine odorant binding protein at acidic pH

Dissociation of bovine odorant binding protein (bOBP) dimers to monomers at pH 2.5 has been confirmed through size exclusion chromatography experiments. Moreover, structural and binding properties of the acidic monomer and neutral dimer have been compared using a combination of experimental (circular dichroism and fluorescence) and computational (molecular dynamics) techniques. The secondary and tertiary structures of bOBP are largely maintained at acidic pH, but molecular dynamics simulations suggest the loop regions (N-terminal residues, Omega-loop and C-terminal segments) are more relaxed and Phe36 and Tyr83 residues are involved in the regulation of the binding cavity entrance. The formation of a molten globule state at acidic pH, suggested by the strong enhancement of fluorescence of 8-anilino-1-naphtalenesulphonic acid (ANS), is not confirmed by any significant change in the near UV circular dichroism spectrum. Functionality measurements, deduced from the interaction of bOBP with 1-amino-anthracene (AMA), show that the binding capacity of the protein at acidic pH is preserved, though slightly looser than at neutral pH. Unfolding of acidic bOBP, induced by guanidinium chloride (GdnHCl), was investigated by means of CD spectroscopy, steady state fluorescence, fluorescence anisotropy and light scattering. The stability of the acidic monomer is lower than that of the neutral dimer, owing to the loss of the swapping interactions, but renaturation is completely reversible. Finally, in contrast with the neutral dimer, at low denaturant concentration some aggregation of the acidic monomer, which vanishes before the unfolding transition, has been observed.     

Glutamate 85 and glutamate 228 contribute to the pH-response of the soluble form of chloride intracellular channel 1

The chloride intracellular channel protein, CLIC1, is synthesised as a soluble monomer that can reversibly bind membranes. Soluble CLIC1 is proposed to respond to the low pH found at a membrane surface by partially unfolding and restructuring into a membrane-competent conformation. This transition is proposed to be controlled by strategically located “pH-sensor” residues that become protonated at acidic pH. In this study, we investigate the role of two conserved glutamate residues, Glu85 in the N-domain and Glu228 in the C-domain, as pH-sensors. E85L and E228L CLIC1 variants were created to reduce pH sensitivity by permanently breaking the bonds these residues form. The structure and stability of each variant was compared to the wild type at both pH 7.0 and pH 5.5. Neither substitution significantly altered the structure but both decreased the conformational stability. Furthermore, E85L CLIC1 formed a urea-induced unfolding intermediate state at both pH 7 and pH 5.5 compared to wild-type and E228L CLIC1 which only formed the intermediate at pH 5.5. We conclude that Glu85 and Glu228 are two of the five pH-sensor residues of CLIC1 and contribute to the pH-response in different ways. Glu228 lowers the stability of the native state at pH 5.5, while Glu85 contributes both to the stability of the native state and to the formation of the intermediate state. By putting these interactions into the context of the three previously described CLIC1 pH-sensor residues, we propose a mechanism for the conversion of CLIC1 from the soluble state to the pre-membrane form.