Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide.

The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S-S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Refolding of cytochromeb 562 and its structural stabilization by introducing a disulfide bond

The packing mechanism of the secondary structures (4-α-helices and 3₁₀-helix) of cytochromeb ₅₆₂ is simulated by the “island model,” where the formation of protein structure is accomplished by the growth-type mechanism with the driving force of packing of the long-range and specific hydrophobic interactions. Packing proceeds through the formation of the structure at the nonhelical part, where a lot of hydrophobic pairs are distributed. Consequently, conformation, nearly similar to the native one, is successfully obtained. With the help of this result, the theoretical prediction of the possibility of forming this disulfide mutant (N22C/G82C) ofb ₅₆₂ can be performed prior to the experiments by our geometrical criterion (“lampshade”). This criterion is expected to be a significant principle for introducing possible disulfide bonds into a protein to be engineered.     

Antigen recognition and targeted delivery by the single-chain Fv

The single-chain Fv (sFv) has proven attractive for immunotargeting, both alone and as a targeting element within sFv fusion proteins. This chapter summarizes the features of sFv proteins that have sparked this interest, starting with the conservation of Fv architecture that makes general sFv design practical. The length and composition of linkers used to bridge V domains are discussed based on the sFv literature; special emphasis is given to the (Gly₄Ser)₃ 15-residue linker that has proven of broad utility for constructing Fv regions of antibodies and other members of the immunoglobulin superfamily. The refolding properties of sFv proteins are summarized and examples given from our laboratory. Spontaneous refolding from the fully reduced and denatured state, typified by 26-10 sFv, is contrasted with disulfide-restricted refolding, exemplified by MOPC 315 and R11D10 sFv proteins, which recover antigen binding only if their disulfides have been oxidized prior to removal of denaturant. The medical value of sFv proteins hinges on their reliability in antigen recognition and rapidity in targeted delivery. Detailed analysis of specificity and affinity of antigen binding by the 26-10 antidigoxin sFv has demonstrated very high fidelity to the binding properties of the parent 26-10 sFv. These results gave confidence to the pursuit of more complex biomedical applications of these proteins, which is indicated by our work with the R11D10 sFv for the imaging of myocardial infarctions. Diagnostic imaging and therapeutic immunotargeting by sFv present significant opportunities, particularly as a result of their pharmacokinetic properties. Intravenously administered sFv offers much faster clearance than conventional Fab fragments or intact immunoglobulin with minimal background binding.     

Refolding of cytochromeb562 and its structural stabilization by introducing a disulfide bond

The packing mechanism of the secondary structures (4--helices and 3₁₀-helix) of cytochromeb ₅₆₂ is simulated by the island model, where the formation of protein structure is accomplished by the growth-type mechanism with the driving force of packing of the long-range and specific hydrophobic interactions. Packing proceeds through the formation of the structure at the nonhelical part, where a lot of hydrophobic pairs are distributed. Consequently, conformation, nearly similar to the native one, is successfully obtained. With the help of this result, the theoretical prediction of the possibility of forming this disulfide mutant (N22C/G82C) ofb ₅₆₂ can be performed prior to the experiments by our geometrical criterion (lampshade). This criterion is expected to be a significant principle for introducing possible disulfide bonds into a protein to be engineered.     

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting

The creation of precise clinical mutations by gene targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.     

Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond.

From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.     

Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the Lewis^Y-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the V_H and V_L domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues V_L100 and V_H44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023–1029, 1995). The B1 disulfide-stabilized Fv (B1dsFv) crystallizes in space group P6₁22 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1-Å resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10-Å wide and 17-Å long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the Lewis^Y tetrasaccharide, Fuc–Gal–Nag–Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781–3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab. Proteins 31:128–138, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Determination of disulfide bond pairs and stability in recombinant tick anticoagulant peptide.

Tick anticoagulant peptide (TAP) is a potent and selective inhibitor of blood coagulation factor Xa (Waxman, L., Smith, D.E., Arcuri, K.E., and Vlasuk, G.P. (1990) Science 248, 593-596). The 60-amino acid sequence of TAP shows limited homology to Kunitz-type inhibitors, including cysteines at positions 5, 15, 33, 39, 55, and 59. For detailed biochemical and pharmacological studies, a recombinant version of TAP (rTAP) has been produced in yeast. To determine the arrangement of the disulfide bonds, rTAP was cleaved with trypsin and chymotrypsin and the purified peptides sequenced using a gas-phase sequenator. The positions of the disulfide bonds were assigned by identifying the cycle(s) at which di-phenylthiohydan-toin-cystine was released. The specific disulfide bridges, Cys-5 to Cys-59, Cys-15 to Cys-39, and Cys-33 to Cys-55, are analogous to those in the prototype Kunitz-type inhibitor, bovine pancreatic trypsin inhibitor (BPTI). While treatment of BPTI with dithiothreitol rapidly and specifically reduced one disulfide bond, the reduction of disulfide bonds in rTAP proceeded at a slower rate and appeared to be nonspecific, reaching a maximum of two disulfides reduced. Reduced rTAP derivatized with either iodoacetic acid or iodoacetamide lost 59% of its inhibitory activity. In contrast, BPTI alkylated with iodoacetic acid inhibited trypsin half as well as the iodoacetamide derivative. Although the arrangement of disulfides in the two inhibitors is the same, their susceptibility to reduction is markedly different.     

The effect of disulfide bond on the conformational stability and catalytic activity of beta-propeller phytase

While beta-propeller phytases (BPPs) from Gram-positive bacteria do not carry disulfide bonding, their counterparts from Gram-negative bacteria contain cysteine residues that may form disulfide bonds. By molecular modeling, two amino acid residues of B. subtilis 168 phytase (168PhyA), Ser-161 and Leu-212, were mutated to cysteine residues. Although the double cysteine mutant was secreted from B. subtilis at an expression level that was 3.5 times higher than that of the wild type, the biochemical and enzymatic properties were unaltered. In CD spectrometric analysis, both enzymes exhibited similar apparent melting temperatures and mid-points of transition under thermal and guanidine hydrochloride induced denaturation, respectively. In enzyme assays, the mutant phytase exhibited a poor refolding ability after thermal denaturation. We postulate that the disulfide bond in BPP sequences from Gram-negative bacteria is beneficial to their stability in the periplasmic compartment. In contrast, the lack of periplasmic space in Bacillus species and the fact that Bacillus BPPs are released extracellularly may render disulfide bonds unnecessary. This may explain why in evolution, BPPs in Bacillus species do not carry disulfide bonds.     

Kinetics of disulfide bond reduction in alpha-lactalbumin by dithiothreitol and molecular basis of superreactivity of the Cys6-Cys120 disulfide bond

Kinetics of disulfide reduction in alpha-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25 degrees C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by bimolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide alpha-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon alpha-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.     

Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T(m)) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution.     

Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.     

Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of proteins. It exists as a covalent dimer in solution, with the 15 kDa monomers linked by an interchain disulfide bond through the Cys101 residues. Sedimentation equilibrium and velocity experiments demonstrated that, after removal of the interchain disulfide bond, GDNF remains as a non-covalent dimer and is stable at pH 7.0. To investigate the effect of the intermolecular disulfide on the structure and stability of GDNF, we compared the solution structures of the wild-type protein and a cysteine-101 to alanine (C101A) mutant using Fourier transform infrared (FTIR), FT-Raman and circular dichroism (CD) spectroscopy and sedimentation analysis. The elimination of the intermolecular disulfide bond causes only minor changes (approximately 4%) in the secondary structures of GDNF. The far- and near-UV CD spectra demonstrated that the secondary and tertiary structures were similar for both wild-type and C101A GDNF. Heparin binding and sedimentation velocity experiments also indicated that the folded structure of the wild-type and C101A GDNF are indistinguishable. The thermal stability of GDNF does not appear to be affected by the absence of the interchain disulfide bond and the biological activity of the C101A mutant is identical with that of the wild-type protein. However, small but significant changes in side chain conformations of tyrosine and aliphatic residues were observed by FT-Raman spectroscopy upon removal of the intermolecular disulfide bond, which may reflect structural changes in the area of dimeric contact. By comparing the Raman spectrum of wild-type GDNF with that of the C101A analog, we identified the conformation of the intermolecular disulfide as trans-gauche-trans geometry. These results indicate that GDNF is an active, properly folded molecule in the absence of the interchain disulfide bond.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG⁰) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.     

Effects of antibody disulfide bond reduction on purification process performance and final drug substance stability

Antibody disulfide bond reduction during monoclonal antibody (mAb) production is a phenomenon that has been attributed to the reducing enzymes from CHO cells acting on the mAb during the harvest process. However, the impact of antibody reduction on the downstream purification process has not been studied. During the production of an IgG₂ mAb, antibody reduction was observed in the harvested cell culture fluid (HCCF), resulting in high fragment levels. In addition, aggregate levels increased during the low pH treatment step in the purification process. A correlation between the level of free thiol in the HCCF (as a result of antibody reduction) and aggregation during the low pH step was established, wherein higher levels of free thiol in the starting sample resulted in increased levels of aggregates during low pH treatment. The elevated levels of free thiol were not reduced over the course of purification, resulting in carry‐over of high free thiol content into the formulated drug substance. When the drug substance with high free thiols was monitored for product degradation at room temperature and 2–8°C, faster rates of aggregation were observed compared to the drug substance generated from HCCF that was purified immediately after harvest. Further, when antibody reduction mitigations (e.g., chilling, aeration, and addition of cystine) were applied, HCCF could be held for an extended period of time while providing the same product quality/stability as material that had been purified immediately after harvest. Biotechnol. Bioeng. 2017;114: 1264–1274. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.     

Effects of introducing negative charges into the molecular surface of thermolysin by site-directed mutagenesis on its activity and stability

Thermolysin is remarkably activated and stabilized by neutral salts, and surface charges are suggested important in its activity and stability. The effects of introducing negative charge into the molecular surface on its activity and stability are described. Seven serine residues were selected, and each of them was changed for aspartate by site-directed mutagenesis in a thermolysin mutant. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide, the k(cat)/K(m) values of all mutants were almost similar to that of the wild-type enzyme (WT). However, those of six out of seven mutants were enhanced 17-19 times with 4 M NaCl, being slightly higher than WT. The remaining casein-hydrolyzing activities of the S53D and S65D mutants (Ser53 and Ser65 are replaced with Asp, respectively) after 30-min incubation with 10 mM CaCl(2) at 85 degrees C were 78 and 63%, being higher than those of WT (51%) and the other mutants (35-53%). S53D was stabilized with increase in the enthalpy change of activation for thermal inactivation while S65D was with decrease in the entropy change of activation. The stability of WT was enhanced by CaCl(2) and reached the level of S53D and S65D at 100 mM, suggesting that S53D and S65D might be stabilized by reinforcement of the Ca(2+)-binding structures.     

Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain

The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110–Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185–Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185–Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.