Model of a LexA repressor dimer bound to recA operator

A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.     

Positional preference of proline in alpha-helices.

Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-(Ala)n-Pro-(Ala)m-NHMe, where n + m = 14, to figure out the positional preference of proline in alpha-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the alpha-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus (i.e., Ncap + 1 position), and the next propensities are found at Ncap, N' (Ncap - 1), and C' (Ccap + 1) positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing alpha-helical conformations, which, however, provides the favored hydration of the corresponding N-H and C=O groups. The highest preference of proline at the beginning of alpha-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of alpha-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of alpha-helix by proline. The average free energy change for the substitution of Ala by Pro in a alpha-helix is computed to be 4.6 kcal/mol, which is in good agreement with the experimental value of approximately 4 kcal/mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme.     

Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.     

Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CA-MA), incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA), has potent antimicrobial activity without toxicity against human erythrocytes. To investigate the effects of the Gly-Ile-Gly hinge sequence of CA-MA on the antibacterial and antitumor activities, two analogues in which the Gly-Ile-Gly sequence of CA-MA is either deleted (P1) or substituted with Pro (P2) were synthesized. The role of the tryptophan residue at position 2 of CA-MA on its antibiotic activity was also investigated using two analogues, in which the Trp2 residue of CA-MA is replaced with either Ala (P3) or Leu (P4). The tertiary structures of CA-MA, P2, and P4 in DPC micelles, as determined by NMR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of alpha-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an alpha-helix from Leu4 to Ala14 without any hinge structure. P1 has significantly decreased lytic activities against bacterial and tumor cells and PC/PS vesicles (3:1, w/w), and reduced pore-forming activity on lipid bilayers, while P2 retained effective lytic activities and pore-forming activity. The N-terminal region of P3 has a flexible structure without any specific secondary structure. The P3 modification caused a drastic decrease in the antibiotic activities, whereas P4, with the hydrophobic Leu side chain at position 2, retained its activities. On the basis of the tertiary structures, antibiotic activities, vesicle-disrupting activities, and pore-forming activities, the structure-function relationships can be summarized as follows. The partial insertion of the Trp2 of CA-MA into the membrane, as well as the electrostatic interactions between the positively charged Lys residues at the N-terminus of the CA-MA and the anionic phospholipid headgroups, leads to the primary binding to the cell membrane. Then, the flexibility or bending potential induced by the Gly-Ile-Gly hinge sequence or the Pro residue in the central part of the peptides may allow the alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA-MA.     

Solution structure of BmP02, a new potassium channel blocker from the venom of the Chinese scorpion Buthus martensi Karsch

BmP02 is a 28-amino acid residue peptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, which had been demonstrated to be a weak blocker of apamin-sensitive calcium-activated potassium channels. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmP02. The results show that BmP02 formed a alpha/beta scorpion fold, the typical three-dimensional structure adopted by most short chain scorpion toxins whose structures have been determined. However, in BmP02 this alpha/beta fold was largely distorted. The alpha-helix was shortened to only one turn, and the loop connecting the helix to the first beta-strand exhibited conformational heterogeneity. The instability of BmP02 could be attributed to a proline at position 17, which is usually a glycine. Because the residue at this position makes intense contact with the alpha-helix, it was supposed that the bulky side chain of proline had pushed the helix away from the beta-sheet. This had a significant influence on the structure and function of BmP02. The alpha-helix rotated by about 40 degrees to avoid Pro17 while forming two disulfides with the second beta-strand. The rotation further caused both ends of the helix to be unwound due to covalent restrictions. According to its structure, BmP02 was supposed to interact with its target via the side chains of Lys11 and Lys13.     

Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.     

Solution structure and cell selectivity of piscidin 1 and its analogues

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure that was isolated from the mast cells of hybrid striped bass [Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268-269]. Pis-1 is not selective for bacterial versus mammalian cells. In the present study, to develop novel antibiotic peptides with selectivity for bacterial cells, we examined the effect of substituting two glycine residues, Gly8 and Gly13, with Ala or Pro on this peptide's structure and biological activities. The bacterial cell selectivity of the peptides decreased in the following order: Gly-->Pro analogues > Gly-->Pro/Ala analogues > Pis-1 > Gly-->Ala analogues. The antimicrobial and hemolytic activities and abilities to permeabilize the model phospholipid membranes were higher for Pis-1 with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13. We determined the tertiary structure of Pis-1 and its analogues in the presence of SDS micelles by NMR spectroscopy. We found that Pis-1 has an alpha-helical structure from Phe2 to Thr21. Also, Pis-1 AA (Gly8, Gly13-->Ala8, Ala13) with higher antibacterial and hemolytic activity than Pis-1 has a stable alpha-helical structure from Phe2 to Thr21. Pis-1 PG (Gly-->Pro8) with bacterial cell selectivity has a hinge structure at Pro8, which provides flexibility in piscidin, followed by a three-turn helix from Val10 to Gly22 in the C-terminal region. Taken together, our results demonstrate that the conformational flexibility provided by introduction of a Pro at position 8, coupled with the primary anchoring of phenylalanines and histidines in the N-terminus to the cell membrane and the optimal length of the C-terminal amphipathic alpha-helix, are the critical factors that confer antibacterial activity and bacterial cell selectivity to Pis-1 PG. Pis-1 PG may be a good candidate for the development of a new drug with potent antibacterial activity but without cytotoxicity.     

Engineering of conformations of plasminogen activator inhibitor-1. A crucial role of beta-strand 5A residues in the transition of active form to latent and substrate forms.

The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface-exposed reactive centre loop (RCL) is inserted as strand 4 in the large central beta-sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximately fourfold increase in the rate of latency transition resulted from His-substitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor template numbering), which interacts with the underlying alpha-helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-helix F and beta-strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of beta-strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes.     

Crystal structures of E. coli CcmG and its mutants reveal key roles of the N-terminal beta-sheet and the fingerprint region

CcmG, also designated DsbE, functions as a periplasmic protein thiol:disulfide oxidoreductase and is required for cytochrome c maturation. Here we report the crystal structures of Escherichia coli CcmG and its two mutants, P144A and the N-terminal fifty seven-residue deletion mutant, and two additional deletion mutants were studied by circular dichroism. Structural comparison of E. coli CcmG with its deletion mutants reveals that the N-terminal beta-sheet is essential for maintaining the folding topology and consequently maintaining the active-site structure of CcmG. Pro144 and Glu145 are key residues of the fingerprint region of CcmG. Pro144 is in cis-configuration, and it makes van der Waals interactions with the active-site disulfide Cys80-Cys83 and forms a C--H...O hydrogen bond with Thr82, helping stabilize the active-site structure. Glu145 forms a salt-bridge and hydrogen-bond network with other residues of the fingerprint region and with Arg158, further stabilizing the active-site structure. The cis-configuration of Pro144 makes the backbone nitrogen and oxygen of Ala143 exposed to solvent, favorable for interacting with binding partners. The key role of cis-Pro144 is verified by the P144A mutant, which contains trans-Ala144 and displays redox property changes. Structural comparison of E. coli CcmG with the recently reported structure of CcmG in complex with the N-terminal domain of DsbD reveals that Tyr141 undergoes conformational changes upon binding DsbD. A cis-proline located at the N-terminus of the first beta-strand of the betabetaalpha motif of the thioredoxin-like domain is a conserved structural feature of the thioredoxin superfamily.     

非解朊栖热菌HG102耐热β-糖苷酶的结构与功能研究

非解朊栖热菌HG10 2耐热 β-糖苷酶为 (β/α)₈桶状结构 ,是具有水解功能和转糖苷功能的单体酶。该酶可以作为一个很好的模型来研究糖苷酶的反应机制、底物特异性和耐热的分子基础。根据对该酶的晶体结构解析和同家族酶的结构比较 ,推测Glu164和Glu338分别是质子供体和亲核基团两个活性位点 ;在α-螺旋N端第一位的脯氨酸和蛋白质外周的精氨酸是耐热机制的关键位点和关键氨基酸残基。为确定这些氨基酸残基的功能 ,通过基因定点突变的方法分别把Glu164、Glu338、Pro316、Pro356、Pro344和Arg325置换成Gln、Ala、Gly、Ala、Phe和Leu ,同时还对Pro316和Pro356进行了双置换。突变酶经过纯化得到电泳纯 ,用CD光谱进行了野生酶和突变酶的结构比较。通过突变酶的酶功能和酶学性质分析 ,结果表明Glu164和Glu338分别是质子供体和亲核基团 ,亲核基团的突变酶TnglyE338A可以合成混合型糖苷键寡糖类似物 ;在α-螺旋N端第一位的Pro316和Pro356以及在蛋白质外周形成离子键的Arg325均是对耐热性有贡献的关键氨基酸残基。     

Mutations in the hinge of a dynamic loop broadly influence functional properties of fructose-1,6-bisphosphatase

Loop 52-72 of porcine fructose-1,6-bisphosphatase may play a central role in the mechanism of catalysis and allosteric inhibition by AMP. The loop pivots between different conformational states about a hinge located at residues 50 and 51. The insertion of proline separately at positions 50 and 51 reduces k(cat) by up to 3-fold, with no effect on the K(m) for fructose 1,6-bisphosphate. The K(a) for Mg(2+) in the Lys(50) --> Pro mutant increases approximately 15-fold, whereas that for the Ala(51) --> Pro mutant is unchanged. Although these mutants retain wild-type binding affinity for AMP and the fluorescent AMP analog 2'(3')-O-(trinitrophenyl)adenosine 5'-monophosphate, the K(i) for AMP increases 8000- and 280-fold in the position 50 and 51 mutants, respectively. In fact, the mutation Lys(50) --> Pro changes the mechanism of AMP inhibition with respect to Mg(2+) from competitive to noncompetitive and abolishes K(+) activation. The K(i) for fructose 2,6-bisphosphate increases approximately 20- and 30-fold in the Lys(50) --> Pro and Ala(51) --> Pro mutants, respectively. Fluorescence from a tryptophan introduced by the mutation of Tyr(57) suggests an altered conformational state for Loop 52-72 due to the proline at position 50. Evidently, the Pro(50) mutant binds AMP with high affinity at the allosteric site, but the mechanism of allosteric regulation of catalysis has been disabled.     

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.     

Investigating the importance of the flexible hinge in caerin 1.1: solution structures and activity of two synthetically modified caerin peptides

Caerin 1.1 is a potent broad-spectrum antibacterial peptide isolated from a number of Australian frogs of the Litoria genus. In membrane-like media, this peptide adopts two alpha-helices, separated by a flexible hinge region bounded by Pro15 and Pro19. Previous studies have suggested that the hinge region is important for effective orientation of the two helices within the bacterial cell membrane, resulting in lysis via the carpet mechanism. To evaluate the importance of the two Pro residues, they were replaced with either Ala or Gly. The antibacterial activity of these two peptides was tested, and their three-dimensional structures were determined using two-dimensional NMR spectroscopy and restrained molecular dynamics calculations. The resulting structures indicate that the central hinge angle decreases significantly upon replacement of the Pro residues with Gly and to a further extent with Ala. This trend was mirrored by a corresponding decrease in antibiotic activity, further exemplifying the necessity of the hinge in caerin 1.1 and related peptides. In a broader context, the use of Pro, Gly, and Ala variants of caerin 1.1 has enabled the relationship between conformational flexibility and activity to be directly investigated in a systematic manner.     

Polyproline II helix conformation in a proline-rich environment: a theoretical study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.     

Proline-induced constraints in alpha-helices

The disrupting effect of a prolyl residue on an α-helix has been analyzed by means of conformational energy computations. In the preferred, nearly α-helical conformations of Ac-Ala₄-Pro-NHMe and of Ac-Ala₇-Pro-Ala₇-NHMe, only the residue preceding Pro is not α-helical, while all other residues can occur in the α-helical A conformation; i.e., it is sufficient to introduce a conformational change of only one residue in order to accommodate proline in a distorted α-helix. Other low-energy conformations exist in which the conformational state of three residues preceding proline is altered considerably; on the other hand, another conformation in which these three residues retain the near-α-helical A-conformational state (with up to 26° changes of their dihedral angles ? and ψ, and a 48° change in one ω from those of the ideal α-helix) has a considerably higher energy. These conclusions are not altered by the substitution of other residues in the place of the Ala preceding Pro. The conformations of the peptide chain next to prolyl residues in or near an α-helix have been analyzed in 58 proteins of known structure, based on published atomic coordinates. Of 331 α-helices, 61 have a Pro at or next to their N-terminus, 21 have a Pro next to their C-terminus, and 30 contain a Pro inside the helix. Of the latter, 16 correspond to a break in the helix, 9 are located inside distorted first turns of the helix, and 5 are parts of irregular helices. Thus, the reported occurrence of prolyl residues next to or inside observed α-helices in proteins is consistent with the computed steric and energetic requirements of prolyl peptides.     

Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

Domain closure mechanism in transferrins: new viewpoints about the hinge structure and motion as deduced from high resolution crystal structures of ovotransferrin N-lobe

The crystal structure of holo hen ovotransferrin N-lobe refined at 1.65 A resolution has been obtained. The final model gave an R-factor of 0.173 in the resolution range between 10.0 and 1.65 A. The comparison of the structure with previous high-resolution apo and Fe(3+)-loaded, domain-opened intermediate structures provides new viewpoints on the domain closure mechanism upon Fe(3+) uptake in ovotransferrin N-lobe. Overall, conformational transition follows the common mechanism that has been first demonstrated for lactoferrin N-lobe; the domains 1 and 2 rotate 49.7 degrees as rigid bodies with a translation of 2.1 A around a screw-axis that passes through the two interdomain beta-strands (89-94 and 244-249). It is generally believed that the two strands display a hinge-like motion. Here, the latter strand indeed displays an ideal hinge nature: the segments 244-246 and 248-249 behave as a part of the rigid body of domain 2 and that of domain 1, respectively, and a sharp bend upon the domain closure is largely accounted for by the changes in the torsion angles phi and psi of Val247. We find, however, that the mode of the conformational change in the first beta-strand is much more complex. Two of the five inter beta-strand hydrogen bonds undergo crucial exchanges: from Ser91-N...Val247-O and Thr89-O...Ala249-N in the open apo and intermediate structures into Tyr92-N...Val247-O and Thr90-O...Ala249-N in the closed holo structure. These exchanges, which may be triggered in the intermediate state by modulation in the topological relation between the Fe(3+)-ligated hinge residue Tyr92-OH and the anion anchor residues of helix 5, are accompanied by a large conformational change and extensive hydrogen bonding rearrangements in a long stretch of segment of Glu82 to Tyr92. Such structural transition would work as a driving force for the domain closure, which highlights a "door closer"-like role, in addition to the canonical-hinge role, for the interdomain polypeptide segment pair. As an alternative hinge that secures the correct domain motion by being placed on a significant distance from the beta-strand hinge, we point out the participation of the van der Waals contacts formed between domain 1 residue of Met331 and domain 2 residues of Trp125, Ile129 and Trp140.     

Design and synthesis of 3alpha-helix peptides forming a cavity for a fluorescent ligand

As a model of receptor protein, a series of 3alpha-helix bundle peptides constructed on a template peptide were designed so as to possess a hydrophobic cavity. The size of cavity was modulated by simple replacements of Leu residues to Ala residues in the hydrophobic core. Binding abilities to 8-anilino-1-naphthalenesulfonic acid (ANS) were estimated by the increase of fluorescence intensity. The peptide having three or four Ala residues in the hydrophobic core remarkably increased the binding ability for ANS, though the peptide having two Ala residues gave an inefficient cavity for ANS. The peptide having six Ala residues decreased the binding ability due to crucial destabilization of the helix bundle structure. This scaffold can be utilized to a receptor model, while further tuning of the sequence is necessary.     

Gating of shaker-type channels requires the flexibility of S6 caused by prolines

The recent crystallization of a voltage-gated K+ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the alpha-helix, both prolines were replaced to promote an alpha-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 alpha-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.