The role of the local environment of engineered Tyr to Trp substitutions for probing the denaturation mechanism of FIS

Factor for inversion stimulation (FIS), a 98-residue homodimeric protein, does not contain tryptophan (Trp) residues but has four tyrosine (Tyr) residues located at positions 38, 51, 69, and 95. The equilibrium denaturation of a P61A mutant of FIS appears to occur via a three-state (N₂ ⇆ I₂ ⇆ 2U) process involving a dimeric intermediate (I₂). Although it was suggested that this intermediate had a denatured C-terminus, direct evidence was lacking. Therefore, three FIS double mutants, P61A/Y38W, P61A/Y69W, and P61A/Y95W were made, and their denaturation was monitored by circular dichroism and Trp fluorescence. Surprisingly, the P61A/Y38W mutant best monitored the N₂ ⇆ I₂ transition, even though Trp38 is buried within the dimer removed from the C-terminus. In addition, although Trp69 is located on the protein surface, the P61A/Y69W FIS mutant exhibited clearly biphasic denaturation curves. In contrast, P61A/Y95W FIS was the least effective in decoupling the two transitions, exhibiting a monophasic fluorescence transition with modest concentration-dependence. When considering the local environment of the Trp residues and the effect of each mutation on protein stability, these results not only confirm that P61A FIS denatures via a dimeric intermediate involving a disrupted C-terminus but also suggest the occurrence of conformational changes near Tyr38. Thus, the P61A mutation appears to compromise the denaturation cooperativity of FIS by failing to propagate stability to those regions involved mostly in intramolecular interactions. Furthermore, our results highlight the challenge of anticipating the optimal location to engineer a Trp residue for investigating the denaturation mechanism of even small proteins.     

Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation

The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.     

Secondary structure extensions in Pyrococcus furiosus ferredoxin destabilize the disulfide bond relative to that in other hyperthermostable ferredoxins. Global consequences for the disulfide orientational heterogeneity.

The single cubane cluster ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) possesses several unique properties when compared even to Fds from other hyperthermophilic archaea or bacteria. These include an equilibrium molecular heterogeneity, a six- to seven-residue increase in size, an Asp rather than the Cys as one cluster ligand, and a readily reducible disulfide bond. NMR assignments and determination of both secondary structure and tertiary contacts remote from the paramagnetic oxidized cluster of Pf 3Fe Fd with an intact disulfide bond reported previously (Teng Q., Zhou, Z. H., Smith, E. T., Busse, S. C., Howard, J. B. Adams, M. W. W., and La Mar, G. (1994) Biochemistry 33, 6316-6328) are extended here to the 4Fe oxidized cluster WT (1H and 15N) and D14C (1H only) Fds with an intact disulfide bond and to the 4Fe oxidized WT Fd (1H and 15N) with a cleaved disulfide bond. All forms are shown to possess a long (13-member) alpha-helix, two beta-sheets (one double-, one triple-stranded), and three turns outside the cluster vicinity, each with tertiary contacts among themselves as found in other Fds. While the same secondary structural elements, with similar tertiary contacts, are found in other hyperthermostable Fds, Pf Fd has two elements, the long helix and the triple-stranded beta-sheet, that exhibit extensions and form multiple tertiary contacts. All Pf Fd forms with an intact disulfide bond exhibit a dynamic equilibrium heterogeneity which is shown to modulate a hydrogen-bonding network in the hydrophobic core that radiates from the Cys21-Cys48 disulfide bond and encompasses residues Lys36, Val24, Cys21, and Cys17 and the majority of the long helix. The heterogeneity is attributed to population of the alternate S and R chiralities of the disulfide bond, each destabilized by steric interactions with the extended alpha-helix. Comparison of the chemical shifts and their temperature gradients reveals that the molecular structure of the protein with the less stable R disulfide resembles that of the Fd with a cleaved disulfide bond. Both cluster architecture (3Fe vs 4Fe) and ligand mutation (Cys for Asp14) leave the disulfide orientational heterogeneity largely unperturbed. It is concluded that the six- to seven-residue extension that results in a longer helix and larger beta-sheet in Pf Fd, relative to other hyperthermostable Fds, more likely serves to destabilize the disulfide bond, and hence make it more readily reducible, than to significantly increase protein thermostability.     

Role of individual disulfide bonds in hen lysozyme early folding steps

To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-alpha-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.     

Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies. Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105. Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies. These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography. Human IL-2 produced in inclusion bodies in E. coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds. Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively. Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.     

An allosteric redox switch in domain V of β2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition

β₂-glycoprotein I (β₂GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that β₂GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β₂GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β₂GPI''s structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β₂GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β₂GPI''s physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.     

Directed evolution of glucose oxidase from Aspergillus niger for ferrocenemethanol-mediated electron transfer

A directed evolution protocol was developed for glucose oxidase (GOx) from Aspergillus niger that mimics applications conditions and employs a well-known mediator, oxidized ferrocenemethanol, in a medium throughput screen (96-well plate format). Upon reduction, oxidized ferrocenemethanol shows a color change from blue to pale yellow that can be recorded at 625 nm. Under optimized screening conditions, a CV of less than 20% was achieved in 96-well microtiter plates. For validating the screening system, two mutant libraries of GOx were generated by standard error-prone PCR conditions (0.04 mM MnCl(2)) and Saccharomyces cerevisiae was employed as host for secreted GOx expression. Two screening of approximately 2000 GOx mutants yielded a double mutant (T30S I94V) with improved pH and thermal resistance. Thermal resistance at a residual activity of 50% was increased from 58 degrees C (wild type, WT) to 62 degrees C (T30S I94V) and pH stability was improved at basic pH (pH 8-11). K(m) for glucose remained nearly unchanged (20.8 mM WT; 21.3 mM T30S I94V) and k(cat) increased (69.5/s WT; 137.7/s T30S I94V).     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A

The eight cysteine residues of ribonuclease A form four disulfide bonds in the native protein. We have analyzed the folding of three double RNase A mutants (C65A/C72A, C58A/C110A, and C26A/C84A, lacking the C65-C72, C58-C110, and C26-C84 disulfide bonds, respectively) and two single mutants (C110A and C26A), in which a single cysteine is replaced with an alanine and the paired cysteine is present in the reduced form. The folding of these mutants was carried out in the presence of oxidized and reduced glutathione, which constitute the main redox agents present within the ER. The use of mass spectrometry in the analysis of the folding processes allowed us (i) to follow the formation of intermediates and thus the pathway of folding of the RNase A mutants, (ii) to quantitate the intermediates that formed, and (iii) to compare the rates of formation of intermediates. By comparison of the folding kinetics of the mutants with that of wild-type RNase A, the contribution of each disulfide bond to the folding process has been evaluated. In particular, we have found that the folding of the C65A/C72A mutant occurs on the same time scale as that of the wild-type protein, thus suggesting that the removal of the C65-C72 disulfide bond has no effect on the kinetics of RNase A folding. Conversely, the C58A/C110A and C26A/C84A mutants fold much more slowly than the wild-type protein. The removal of the C58-C110 and C26-C84 disulfide bonds has a dramatic effect on the kinetics of RNase A folding. Results described in this paper provide specific information about conformational folding events in the regions involving the mutated cysteine residues, thus contributing to a better understanding of the complex mechanism of oxidative folding.     

Role of disulfide bonds in conformational stability and folding of 5'-deoxy-5'-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5'-deoxy-5'-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138-C205 and C200-C262) and a CXC motif (C259-C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase

During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used. Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl. On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea). However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl. Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa). Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt. Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state. Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase. Refolding from this state only provided low recoveries of native enzyme.     

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.     

Disulfide bonds of phospholipase A2 from bee venom yield discrete contributions to its conformational stability

Disulfide bonds are known to be crucial for protein stability. To probe the contribution of each of the five disulfide bonds (C9-C31, C30-C70, C37-C63, C61-C95, and C105-C113) in bee venom phospholipase A₂ to stability, variants with deleted disulfide bonds were produced by substituting two serine residues for each pair of cysteine residues. The mutations started from the pseudo-wild-type variant (pWT) with the mutation I1A (Markert et al., Biotechnol. Bioeng. 98 (2007) 48-59). All variants were expressed in Escherichia coli, refolded from inclusion bodies and purified as pWT. The activity of the variants ranged from 12 to 82% of pWT. From the transition curves of guanidine hydrochloride-induced unfolding, the contributions of the individual disulfide bonds to conformational stability were estimated. They increased in the sequence C9-C31 < C105-C113 < C30-C70 ≈ C37-C63 < C61-C95. For two disulfide bonds (C9-C31, C105-C113) the effects were confirmed on additionally produced variants with the substitution of cysteine by alanine. Despite distinct differences in stability, all variants showed similar cooperativity in unfolding. Selected variants were also probed for proteolytic stability toward thermolysin. The removal of disulfide bonds increased the proteolytic susceptibility of the native proteins in the same way as the stability decreased. From the comparison of the results with literature data on phospholipase A₂ from bovine pancreas possessing seven disulfide bonds, it was concluded that conserved disulfide bonds in homologous proteins fulfill related functions in conformational stability.     

Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.     

A truncated peptide model of the mutant P61A FIS forms a stable dimer

Factor for inversion stimulation (FIS) is a 98-residue homodimeric DNA-binding protein involved in several different cellular processes including DNA inversion and the regulation of multiple genes. FIS contains a flexible and functionally important N-terminus followed by four helices (A-D), the last two of which consist of the DNA-binding region. Helix B, which comprises the main dimerization interface has a 20 degrees kink at its center that was originally thought to be caused by the presence of a proline at position 61. However, it was later shown that the kink remained largely intact and that FIS retained its native-like function when the proline was mutated to an alanine. We previously showed that the P61A mutation increased the stability of FIS, but decreased its equilibrium denaturation cooperativity apparently due to preferential stabilization of the B-helix. Here we studied a peptide of P61A FIS, corresponding to residues 26-71 (26-71(A3) FIS), which encompasses the dimer interface (helices A and B). Circular dichroism (CD) and size-exclusion chromatography/multi-angle light scattering showed that the peptide was alpha-helical and dimeric, respectively, as expected based on the 3D structure of FIS. Urea-induced equilibrium denaturation experiments monitored by far-UV CD revealed a concentration-dependent transition, and data analysis based on a N2<-->2U model yielded a DeltaG of approximately -10 kcal/mol. Our results suggest that 26-71(A3) FIS can form a stable dimeric structure despite lacking the N- and C-terminus of native FIS.     

Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.     

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.     

Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.