Substitution Of Hydrophobic Residues Research Articles

A single amino acid residue tunes the stability of the fully reduced flavin cofactor and photorepair activity in photolyases

The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes.

Discovery of a Novel Antimicrobial Peptide, Temporin-PKE, from the Skin Secretion of Pelophylax kl. esculentus, and Evaluation of Its Structure-Activity Relationships.

Bacterial resistance against antibiotics has led to increasing numbers of treatment failures, and AMPs are widely accepted as becoming potential alternatives due to their advantages. Temporin-PKE is a novel peptide extracted from the skin secretion of Pelophylax kl. esculentus and it displays a strong activity against Gram-positive bacteria, with an extreme cytotoxicity. Incorporating positively charged residues and introducing D-amino acids were the two main strategies adopted for the modifications. The transformation of the chirality of Ile could reduce haemolytic activity, and an analogue with appropriate D-isoforms could maintain antimicrobial activity and stability. The substitution of hydrophobic residues could bring about more potent and broad-spectrum antimicrobial activities. The analogues with Lys were less harmful to the normal cells and their stabilities remained at similarly high levels compared to temporin-PKE. The optimal number of charges was three, and the replacement on the polar face was a better choice. Temporin-PKE-3K exerted dually efficient functions includingstrong antimicrobial and anticancer activity. This analogue showed a reduced possibility for inducing resistance in MRSA and Klebsiella pneumoniae, a rather strong antimicrobial activity in vivo, and it exhibited the highest therapeutic index such that temporin-PKE-3K has the potential to be developed as a clinical drug.

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Activity enhancement of CotA laccase by hydrophilic engineering, histidine tag optimization and static culture.

CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids. Among them, the protein yield of the mutant F207Y was increased by 30%; the catalytic activity (kcat/Km) of V403T and P455S was two and three times higher than that of CH6-CotA, respectively. Finally, triple mutant F2071Y/V403T/P455S with C-terminal his-tag (CH6-TSY) was constructed. When the proteins were expressed in microanaerobic condition, the activities of mutants CH6-P455S and CH6-TSY were enhanced about 48- and 42-folds compared to that of NH6-CotA in non-static culture.

Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155-1168]. Here, we show that the substitution of hydrophobic residues-specifically, Y69, F95, F96, and W203-with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar-polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.

Design, synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC50=53.02nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.

Improved thermostability of esterase from Aspergillus fumigatus by site-directed mutagenesis

A 1.020-bp esterase gene, estQ, encoding for a protein of 339 amino acids, was cloned from Aspergillus fumigatus and expressed in E. coli. EstQ exhibited the optimal activity around 40°C and pH 9.0. In order to obtain more thermostable esterases, three mutants (A134T, V160T, A134T–V160T) were constructed by site-directed mutagenesis and also characterized for further research. Compared to A134T and V160T displaying their optimum activity at 40°C, A134T–V160T exhibited a 5°C higher optimal temperature and a longer half-life more than 24 times than that of WT at 50°C. All the mutants displayed favorable effects on thermostability and retained 53–76% activity after pre-incubation for 30min at 45°C, about 20–40% higher than that of the WT. With an increase in Km of the three mutants, a decrease in catalytic efficiency in kcat/Km was observed in mutant V160T and A134T–V160T against p-nitrophenyl butyrate. Homology models of WT and A134T–V160T were built to understand the structure–function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction and additional hydrogen bonds formed in the mutants by substitution of hydrophobic residues with hydrophilic residues. This study provide useful theoretical reference for enzyme evolution in vitro.

Mechanism of action and specificity of antimicrobial peptides designed based on buforin IIb

Buforin IIb-a synthetic analog of buforin II that contains a proline hinge between the two α-helices and a model α-helical sequence at the C-terminus (3× RLLR)-is a potent cell-penetrating antimicrobial peptide. To develop novel antimicrobial peptides with enhanced activities and specificity/therapeutic index, we designed several analogs (Buf III analogs) by substitutions of amino acids in the proline hinge region and two α-helices of buforin IIb, and examined their antimicrobial activity and mechanism of action. The substitution of hydrophobic residues ([F6] and [V8]) in the proline hinge region with other hydrophobic residues ([W6] and [I8]) did not affect antimicrobial activity, while the substitution of the first four amino acids RAGL with a model α-helical sequence increased the antimicrobial activity up to 2-fold. Like buforin IIb, Buf III analogs penetrated the bacterial cell membranes without significantly permeabilizing them and were accumulated inside Escherichia coli. Buf III analogs were shown to bind DNA in vitro and the DNA binding affinity of the peptides correlated linearly with their antimicrobial potency. Among the Buf III analogs, the therapeutic index of Buf IIIb and IIIc (RVVRQWPIG[RVVR]3 and KLLKQWPIG[KLLK]3, respectively) were improved 7-fold compared to that of buforin IIb. These results indicate that Buf III analogs appear to be promising candidates for future development as novel antimicrobial agents.

Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: importance of residues in the orotate binding site.

The reaction catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) is accompanied by exceptional values for rate enhancement (k(cat)/k(non) = 7.1 × 10(16)) and catalytic proficiency [(k(cat)/K(M))/k(non) = 4.8 × 10(22) M(-1)]. Although a stabilized vinyl carbanion/carbene intermediate is located on the reaction coordinate, the structural strategies by which the reduction in the activation energy barrier is realized remain incompletely understood. This laboratory recently reported that "substrate destabilization" by Asp 70 in the OMPDC from Methanothermobacter thermoautotrophicus (MtOMPDC) lowers the activation energy barrier by ∼5 kcal/mol (contributing ~2.7 × 10(3) to the rate enhancement) [Chan, K. K., Wood, B. M., Fedorov, A. A., Fedorov, E. V., Imker, H. J., Amyes, T. L., Richard, J. P., Almo, S. C., and Gerlt, J. A. (2009) Biochemistry 48, 5518-5531]. We now report that substitutions of hydrophobic residues in a pocket proximal to the carboxylate group of the substrate (Ile 96, Leu 123, and Val 155) with neutral hydrophilic residues decrease the value of k(cat) by as much as 400-fold but have a minimal effect on the value of k(ex) for exchange of H6 of the FUMP product analogue with solvent deuterium; we hypothesize that this pocket destabilizes the substrate by preventing hydration of the substrate carboxylate group. We also report that substitutions of Ser 127 that is proximal to O4 of the orotate ring decrease the value of k(cat)/K(M), with the S127P substitution that eliminates hydrogen bonding interactions with O4 producing a 2.5 × 10(6)-fold reduction; this effect is consistent with delocalization of the negative charge of the carbanionic intermediate on O4 that produces an anionic carbene intermediate and thereby provides a structural strategy for stabilization of the intermediate. These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis.

Folding Intermediate and Folding Nucleus for I→N and U→I→N Transitions in Apomyoglobin: Contributions by Conserved and Nonconserved Residues

Kinetic investigation on the wild-type apomyoglobin and its 12 mutants with substitutions of hydrophobic residues by Ala was performed using stopped-flow fluorescence. Characteristics of the kinetic intermediate I and the folding nucleus were derived solely from kinetic data, namely, the slow-phase folding rate constants and the burst-phase amplitudes of Trp fluorescence intensity. This allowed us to pioneer the ϕ-analysis for apomyoglobin. As shown, these mutations drastically destabilized the native state N and produced minor (for conserved residues of G, H helices) or even negligible (for nonconserved residues of B, C, D, E helices) destabilizing effect on the state I. On the other hand, conserved residues of A, G, H helices made a smaller contribution to stability of the folding nucleus at the rate-limiting I→N transition than nonconserved residues of B, D, E helices. Thus, conserved side chains of the A-, G-, H-residues become involved in the folding nucleus before crossing the main barrier, whereas nonconserved side chains of the B-, D-, E-residues join the nucleus in the course of the I→N transition.

N- and C-Terminal Hydrophobic Patches Are Involved in Fibrillation of Glucagon

Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.

Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly

Lipoprotein(a) [Lp(a)] is assembled via an initial noncovalent interaction between apolipoprotein B100 (apoB) and apolipoprotein(a) [apo(a)] that facilitates the formation of a disulfide bond between the two proteins. We previously reported that a lysine-rich, alpha-helical peptide spanning human apoB amino acids 4372-4392 was an effective inhibitor of Lp(a) assembly in vitro. To identify the important structural features required for inhibitory action, new variants of the apoB4372-4392 peptide were investigated. Introduction of a central leucine to proline substitution abolished the alpha-helical structure of the peptide and disrupted apo(a) binding and inhibition of Lp(a) formation. Substitution of hydrophobic residues in the apoB4372-4392 peptide disrupted apo(a) binding and inhibition of Lp(a) assembly without disrupting the alpha-helical structure. Substitution of all four lysine residues in the peptide with arginine decreased the IC50 from 40 microM to 5 microM . Complexing of the arginine-substituted peptide to dimyristoylphosphatidylcholine improved its activity further, yielding an IC50 of 1 microM. We conclude that the alpha-helical structure of apoB4372-4392, in combination with hydrophobic residues at the lipid/water interface, is crucial for its interaction with apo(a). Furthermore, the interaction of apoB4372-4392 with apo(a) is not lysine specific, because substitutions with arginine result in a more effective inhibitor.

Mapping the electron transfer interface between cytochrome b5 and cytochrome c.

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.

Features in the N and C Termini of the MAPK-interacting Kinase Mnk1 Mediate Its Nucleocytoplasmic Shuttling

Eukaryotic initiation factor eIF4E binds to the 5'-cap structure of the mRNA and also to the molecular scaffold protein eIF4G. eIF4E is a phosphoprotein, and the kinases that act on it have been identified as the MAPK-interacting kinases Mnk1 and Mnk2. Mnk1/2 also bind to the scaffold protein eIF4G. The N-terminal region of Mnk1 has previously been shown to bind to importin alpha, a component of the nuclear transport machinery, although Mnk1 itself is cytoplasmic. Here we identify a CRM1-type nuclear export motif in the C-terminal part of Mnk1. Substitution of hydrophobic residues in this motif results in Mnk1 becoming nuclear. This has allowed us to study the features of Mnk1 that are involved in its transport to the nucleus. This process requires part, but not all, of a polybasic region near the N terminus of Mnk1. Residues required for nuclear transport are also required for its interaction with importin alpha. This polybasic region also serves a second function in that it is required for the binding of Mnk1 to eIF4G, although the residues involved in this interaction are not identical to those involved in the binding of Mnk1 to importin alpha. Interaction of Mnk1 with eIF4G promotes the phosphorylation of eIF4E. Mutations that reduce the binding of Mnk1 to eIF4G in vivo and in vitro also decrease the ability of Mnk1 to enhance eIF4E phosphorylation in vivo, underlining the importance of the eIF4G-Mnk1 interaction in this process.

Effects of denaturants and substitutions of hydrophobic residues on backbone dynamics of denatured staphylococcal nuclease.

Analysis of residual dipolar couplings (RDCs) in the Delta131Delta fragment of staphylococcal nuclease has demonstrated that its ensemble-averaged structure is resistant to perturbations such as high concentrations of urea, low pH, and substitution of hydrophobic residues, suggesting that its residual structure is encoded by local side-chain/backbone interactions. In the present study, the effects of these same perturbations on the backbone dynamics of Delta131Delta were examined through (1)H-(15)N relaxation methods. Unlike the global structure reported by RDCs, the transverse relaxation rates R(2) were quite sensitive to denaturing conditions. At pH 5.2, Delta131Delta exhibits an uneven R(2) profile with several characteristic peaks involving hydrophobic chain segments. Protonation of carboxyl side chains by lowering the pH reduces the values of R(2) along the entire chain, yet these characteristic peaks remain. In contrast, high concentrations of urea or the substitution of 10 hydrophobic residues eliminates these peaks and reduces the R(2) values by a greater amount. The combination of low pH and high urea leads to further decreases in R(2). These denaturant-induced increases in backbone mobility are also reflected in decreases in (15)N NOEs and in relaxation interference parameters, with the former reporting an increase in fast motions and the latter a decrease in slow motions. Comparison between the changes in chain dynamics and the corresponding changes in Stokes radius and the patterns of RDCs suggests that regional variations in backbone dynamics in denatured nuclease arise primarily from local contacts between hydrophobic side chains and local interactions involving charged carboxyl groups.

In vivo studies of HDL assembly and metabolism using adenovirus-mediated transfer of ApoA-I mutants in ApoA-I-deficient mice.

We have used adenovirus-mediated gene transfer in apoA-I-deficient (A-I-/-) mice to probe the in vivo assembly and metabolism of HDL using apoA-I variants, focusing primarily on the role of the C-terminal 32 amino acids (helices 9-10). Lipid, lipoprotein, and apoA-I analyses showed that plasma levels of apoA-I and HDL of the mutants were 40-88% lower than that of wild type (WT) human apoA-I despite comparable levels of expression in the liver. WT apoA-I and mutant 1 (P165A, E172A) formed spherical particles with the size and density of HDL2 and HDL3. Mutant 2 (E234A, E235A, K238A, K239A) generated spherical particles with density between HDL2 and HDL3. Mutant 3 (L211V, L214V, L218V, L219V) and mutant 4 (L222K, F225K, F229K), which have substitutions of hydrophobic residues in the C-terminus, generated discoidal HDL particles indicating a defect in their conversion to mature spherical HDL. Significant amounts of mutant 4 and mutant 5 (truncated at residue 219) were found in the lipid poor fractions after ultracentrifugation of the plasma (18 and 35%, respectively, of total apoA-I). These findings suggest that hydrophobic residues in and/or between helices 9 and 10 are important for the maturation of HDL in vivo.

Substitution of surface-exposed framework residues alters secretion of recombinant fusion protein Fv/tumor necrosis factor in Escherichia coli.

Substitution of hydrophobic residues with hydrophilic ones at surface-exposed positions may influence the yield of antibody fragment expression in Escherichia coli by reducing its aggregation potential. We introduced such substitutions at 8 surface-exposed framework region residues of a fusion protein Fv/Tumor Necrosis Factor, which resulted in the expression of 10 mutant fusion proteins (Mut 1-10) in E. coli. Our results showed that expression levels of Mut 1-3, with mutations of A9S, T18K, and G41D, respectively, decreased by 4- to 8-fold, whereas expression levels of Mut 4, 9, and 10, with mutations of S60D/S70D, T28D, and G65D, respectively, were not affected. In contrast, mutation of F83A at light-chain residue 83, which is usually buried at the variable/constant domain interface of antibody molecules but becomes surface-exposed in recombinant Fv molecules, increased the expression level of Mut 5 by 4-fold. Our results suggest that this important substitution of a hydrophobic residue with a hydrophilic one may also be applied to increase the secretion of other recombinant Fv molecules in E. coli periplasm.

Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states.

1. Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K+ channels induced by the removal of extracellular potassium, K+o. 2. In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3. To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mM K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4. The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild-type Kv1.3 channel. 5. CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6. We conclude that removal of K+o induces the Kv1.3 channel to transition to a non-conducting 'closed' state which can switch into a non-conducting 'inactivated' state upon depolarization.

The Cu,Zn superoxide dismutase from Escherichia coli retains monomeric structure at high protein concentration. Evidence for altered subunit interaction in all the bacteriocupreins.

Gel-filtration chromatography experiments performed at high protein concentrations demonstrate that the Cu,Zn superoxide dismutase from Escherichia coli is monomeric irrespective of the buffer and of ionic strength. The catalytic activity of the recombinant enzyme is comparable with that of eukaryotic isoenzymes, indicating that the dimeric structure commonly found in Cu,Zn superoxide dismutases is not necessary to ensure efficient catalysis. The analysis of the amino acid sequences suggests that an altered interaction between subunits occurs in all bacterial Cu,Zn superoxide dismutases. The substitution of hydrophobic residues with charged ones at positions located at the dimer interface of all known Cu,Zn superoxide dismutases could be specifically responsible for the monomeric structure of the E. coli enzyme.

Role of interchain alpha-helical hydrophobic interactions in Ca2+ affinity, formation, and stability of a two-site domain in troponin C.

We have previously shown that a 34-residue synthetic peptide representing the calcium-binding site III of troponin C formed a symmetric two-site dimer consisting of two helix-loop-helix motifs arranged in a head-to-tail fashion (Shaw, G.S., Hodges, R.S., & Sykes, B.D., 1990, Science 249, 280-283). In this study the hydrophobicities of the alpha-helices were altered by replacing L-98 and F-102 in the N-terminal region and/or I-121 and L-122 in the C-terminal region with alanine residues. Our results showed that substitution of hydrophobic residues either in the N- or C-terminal region have little effect on alpha-helix formation but resulted in a 100- and 300-fold decrease in Ca2+ affinity, respectively. Simultaneous substitution of both hydrophobes in the N- and C-terminal region resulted in a 1,000-fold decrease in Ca2+ affinity. Data from guanidine hydrochloride denaturation studies suggested that intermolecular interactions occur and that the less hydrophobic analogs had a lower overall conformational stability. These data support the contention that the hydrophobic residues are important in the formation of the two-site domain in troponin C, and this hydrophobic association stabilizes Ca2+ affinity.