Hetero-alpha-helical, two-chain, coiled coils: alpha beta hybrid tropomyosin

The nature of the interhelix interaction in two-chain, α-helical, coiled coils is studied by experiments on the formation of hydrid molecules in which one helical chain is an α-tropomyosin and the other a β-tropomoysin. By means of a recently developed assay, the population of heterohelical (i.e., αβ hybrid) molecules relative to their homohelical (αα and ββ) parent species is determined under a variety of conditions, both equilibrium and nonequilibrium. It is found that mixed intact αα and ββ molecules do not form hybrid species in detectible amounts even after incubation at room temperature (or below) for periods of over one week. That the lack of αβ species in this “native-exchange” system is a result of a kinetic barrier is evident from experiments involving a thermal denaturation–renaturation cycle in which the largely dissociated, unfolded chains at higher temperature are annealed to benign temperatures over a period of 6 h, thus assuring an equilibrium population of two-chain species. In the resulting equilibrium state, the αβ population is one-half the total, indicating that recombination is random. Furthermore, this same (equilibrium) state is reached if the separated, mostly unfolded chains are renatured by a rapid (～ 40 s) quench to benign temperatures. Some implications of these results for the thermodynamics of interhelix interation, for kinetics of chain dissociation and recombination, and for in vivo genesis of two-chain coiled coils are discussed.     

Lignans from Mosla scabra

Two new cyclobutane-type lignans, named moslolignans A and B, together with two known ones, andamanicin and magnosalin, were isolated from the whole plant of Mosla scabra. Their structures were established as 1beta*,2beta*,3alpha*,4alpha*-1,2-dimethyl-3-(3- methoxy-4,5-methylene-dioxyphenyl)-4-(2,4,5-trimethoxyphenyl)-cycl obutane and 1beta*,2beta*,3alpha*,4alpha*-1,2-dimethyl-3-(2, 5-dimethoxy-3,4-methylenedioxyphenyl)-4-(2,4,5-trimethoxyphenyl)-cyclobu tane by spectroscopic methods. This is the first report of naturally-occurring cyclobutane-type lignans with asymmetrical substitutions.     

bis-Azaaromatic quaternary ammonium analogues: ligands for alpha4beta2* and alpha7* subtypes of neuronal nicotinic receptors

A series of bis-nicotinium, bis-pyridinium, bis-picolinium, bis-quinolinium and bis-isoquinolinium compounds was evaluated for their binding affinity at nicotinic acetylcholine receptors (nAChRs) using rat brain membranes. N,N'-Decane-1,12-diyl-bis-nicotinium diiodide (bNDI) exhibited the highest affinity for [(3)H]nicotine binding sites (K(i)=330 nM), but did not inhibit [(3)H]methyllycaconitine binding (K(i) >100 microM), indicative of an interaction with alpha4beta2*, but not alpha7* receptor subtypes, respectively. Also, bNDI inhibited (IC(50)=3.76 microM) nicotine-evoked (86)Rb(+) efflux from rat thalamic synaptosomes, indicating antagonist activity at alpha4beta2* nAChRs. N,N'-Dodecane-1,12-diyl-bis-quinolinium dibromide (bQDDB) exhibited highest affinity for [(3)H]methyllycaconitine binding sites (K(i)=1.61 microM), but did not inhibit [(3)H]nicotine binding (K(i)>100 microM), demonstrating an interaction with alpha7*, but not alpha4beta2* nAChRs. Thus, variation of N-n-alkyl chain length together with structural modification of the azaaromatic quaternary ammonium moiety afforded selective antagonists for the alpha4beta2* nAChR subtype, as well as ligands with selectivity at alpha7* nAChRs.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix.

The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.     

Kinetics of folding and unfolding of beta beta-tropomyosin.

The kinetics of folding from random coils to two-chain coiled coils of beta beta-tropomyosin was studied by stopped-flow CD (SFCD) in the backbone region (222 nm). Two species were studied: the reduced form and the doubly disulfide cross-linked form. The proteins were totally unfolded in 6M urea-saline buffer, then refolded by tenfold dilution into benign buffer. In the refolding medium, they spontaneously recover the two-chain coiled-coil structure. Reduced beta beta refolds in at least two stages: one or more fast phases (< 0.04 s), in which an intermediate with 71% of the equilibrium ellipticity forms, followed by a slower time-resolvable phase that completes the folding. The slow phase is first order, signifying that dimerization occurs in the fast phase. The time constant of the slow phase is 2 s at 20 degrees C and requires activation parameters of delta S not equal to = -7 +/- 0.3 cal/mol.K, delta H not equal to = 15 +/- 1 kcal/mol. These results are very similar to those previously found for the reduced genetic variant alpha alpha-tropomyosin. In contrast, refolding of doubly disulfide cross-linked beta beta is complete within the dead time (< 0.04 s), whereas the singly cross-linked alpha alpha species also displays a slow phase. The opposite process, unfolding reduced beta beta from the coiled-coil state, is complete within the dead time, as in the alpha alpha variant.     

Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.     

Kinetic studies on partially liganded species of carboxyhemoglobin: alpha 2 CO beta 2 and alpha 2 beta 2CO

The valency hybrids of Hb A, alpha 2CO beta 2+, and alpha 2+ beta 2CO have been prepared by a new high pressure liquid chromatography method, and the kinetics of their CO-combination and dissociation reactions have been studied by double mixing and microperoxidase methods. Both reactions are biphasic. The slow phase in CO-combination and the fast phase in CO-dissociation are due to the reactions of alpha CO2 beta T2 or alpha 2 beta 2CO,T. The fast phase in CO-combination reaction has two components, one due to the dimers of the hybrid and the other due to the R-state tetramer. Immediately after the reduction of the valency hybrids, the overall system is represented by the equation: 2 alpha CO beta in equilibrium alpha 2CO beta 2R in equilibrium alpha 2CO beta 2T or (formula: see text) If the solutions are aged for 3-11 s, the R-state population is reduced gradually to a very small size, and the main species after 11 s of aging are dimers and T-state tetramers. Analysis of the kinetic data indicates slow R in equilibrium T equilibria in the absence of phosphates and significant dissociation of the T-state tetramer. It is concluded that the subunit contacts alpha 1-beta 2 (or alpha 2-beta 1) are impaired seriously in the hybrids. Very slow R in equilibrium T relaxation makes these hybrids unlikely intermediates in the sequential binding of CO to Hb tetramer.     

Effect of phosphate on the kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains

The kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains was investigated under various buffer conditions by use of a circular dichroism (CD) stopped-flow apparatus. The difference CD spectra of hemoglobin against its constituent chains were independent of the buffer conditions, while the time courses of the Soret CD after mixing equimolar amounts of the alpha and beta chains changed with the buffer conditions. The time courses were analyzed on the basis of a scheme which included a monomer-tetramer equilibrium of the beta chain (beta 4 in equilibrium 4 beta), dissociation of the beta 4 (beta 4 leads to 4 beta), and a second-order combination of alpha and beta monomers (alpha + beta leads to alpha beta). The analysis showed that buffer conditions affected the dissociation of the beta 4 rather than the monomer combination: The rate of the dissociation of the beta 4 accelerated with decreasing phosphate concentration, while the rate of the monomer combination was less sensitive to the phosphate concentration. This result indicates that the stability of the beta 4 depends on the phosphate concentration. It was furthermore suggested that the inorganic phosphate was bound to the beta 4 with an association constant of 133 M-1 and a Hill coefficient of 1.2.     

Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.

The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).     

On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.     

Alpha-helix to random-coil transition of two-chain, coiled coils: experiments on the thermal denaturation of doubly cross-linked beta beta tropomyosin

Equilibrium thermal denaturation curves (by circular dichroism) are reported for doubly cross-linked beta beta tropomyosin two-chain coiled coils. Cross-linking was performed by reaction of sulfhydryls with either ferricyanide or 5,5'-dithiobis(2-nitrobenzoate) (NbS2). The extent of reaction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and either by titration of residual sulfhydryls with NbS2 (ferricyanide cross-linking) or by determination of mixed disulfide (protein-S-SbN) through reaction with dithiothreitol (NbS2 cross-linking). The results indicate approximately 90% conversion to molecules with interchain cross-links at both C-36 and C-190. Thermal unfolding curves are compared with those obtained previously for non-cross-linked species. The curves are indistinguishable up to approximately 40 degrees C. Above approximately 40 degrees C, the doubly cross-linked species is more stable, but the transition is less steep. This relationship is also compared with that found between alpha alpha tropomyosin (a similar coiled coil made of a genetic variant chain having a sulfhydryl only at C-190) and its singly cross-linked derivative. Thermal curves for alpha alpha and beta beta non-cross-linked species are very similar, alpha alpha being somewhat more stable. For cross-linked alpha alpha, however, the curve sags at temperatures somewhat below the region of principal cooperative loss of helix, the latter occurring at higher temperature but with the same steepness as in the non-cross-linked case. The sag has been ascribed to a "pretransition" in the region of C-190. Thus, doubly and singly cross-linked species differ in that the former show no pretransition and decreased steepness in the principal transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

A scanning calorimetric study of the thermally induced unfolding of various forms of tropomyosin

The reversible thermally induced unfolding of various forms of tropomyosin, a two-chain alpha-helical coiled coil, has been studied by high-sensitivity differential scanning calorimetry (DSC). Included in the study are the reduced and oxidized (disulfide cross-linked) forms of alpha alpha- and beta beta-tropomyosin, and the forms of alpha alpha-tropomyosin in which all sulfhydryl groups have been blocked by carboxymethylation or carboxyamidomethylation. Oxidation or blocking of the sulfhydryl groups of tropomyosin strongly affect the thermotropic behavior of the protein in unpredictable ways. The empirical results presented here are in qualitative agreement with those from an earlier DSC study of the oxidized and carboxymethylated forms of alpha alpha-tropomyosin [S.A. Potekhin and P.L. Privalov (1982) Journal of Molecular Biology, Vol. 159, pp. 519-535], but we find that a different decomposition into subtransitions is possible. Comparison of the alpha alpha and beta beta species indicates, in agreement with extant CD studies, that the noncross-linked beta beta species is somewhat less stable than its alpha alpha counterpart, but that cross-linking enhances the stability of the beta beta doubly cross-linked species by a greater amount and does not lead to the small low-temperature transition ("pretransition") seen in the singly cross-linked alpha alpha species.     

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unfolding/refolding studies of smooth muscle tropomyosin. Evidence for a chain exchange mechanism in the preferential assembly of the native heterodimer

The thermal and the urea-induced unfolding profiles of the coiled-coil alpha-helix of native and refolded tropomyosin from chicken gizzard were studied by circular dichroism. Refolding of tropomyosin at low temperature from alpha + beta subunits, dissociated by guanidinium chloride, urea, or high temperature, predominantly produced alpha alpha + beta beta homodimers in agreement with earlier studies of refolding from guanidinium chloride (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The presence of two unfolding transitions in low salt solutions with about equal helix loss verified the composition with the first unfolding transition of the homodimer mixture originating from alpha alpha. In contrast, refolding by equilibrating at temperatures close to physiological, however, produced the native alpha beta heterodimer, which unfolded in a single transition. The refolding kinetics of dissociated alpha + beta subunits indicated that beta beta homodimers form first, leading to alpha alpha homodimers both of which are relatively stable against chain exchange below approximately 25 degrees C. Equilibrating the homodimer mixture at 37-40 degrees C for long times, however, produced the native alpha beta molecule via chain exchange. The equilibria involved indicate that the free energy of formation from subunits of alpha beta is much less than that of (alpha alpha + beta beta)/2. In vivo folding of alpha beta from the two separate alpha and beta gene products is, therefore, thermodynamically favored over the formation of homodimers and biological factors need not be considered to explain the native preferred alpha beta composition.     

Lipid--protein multiple binding equilibria in membranes

Phospholipids at the lipid--protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on two integral membrane proteins, cytochrome oxidase and (Na,-K)-ATPase, reconstituted into defined phospholipids. Electron spin resonance measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K congruent to 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the second experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per alpha 2 beta 2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.     

Nuclear Overhauser effect and cross-relaxation rate determinations of dihedral and transannular interproton distances in the decapeptide tyrocidine A.

The following interproton distances are reported for the decapeptide tyrocidine A in solution: (a) r(phi) distances between NH(i) and H alpha (i), (b) r(psi) distances between NH (i + 1) and H alpha (i), (c) r(phi psi) distances between NH(i + 1) and NH(i), (d) NH in equilibrium NH transannular distances, (e) H alpha in equilibrium H alpha transannular distances, (f) r x 1 distances between H alpha and H beta protons, (g) NH(i) in equilibrium H beta (i) distances, (h) NH (i + 1) in equilibrium H beta (i) distances, (i) carboxamide-backbone protons and carboxamide-side chain proton distances, (j) side chain proton-side chain proton distances. The procedures for distance calculations were: NOE ratios and calibration distances, sigma ratios and calibration distances, and correlation times and sigma parameters. The cross-relaxation parameters were obtained from the product, say, of NOE 1 leads to 2 and the monoselective relaxation rate of proton 2; the NOEs were measured by NOE difference spectroscopy. The data are consistent with a type I beta-turn/ type II' beta-turn/ approximately antiparallel beta-pleated sheet conformation of tyrocidine A in solution and the NOEs, cross-relaxation parameters, and interproton distances serve as distinguishing criteria for beta-turn and beta-pleated sheet conformations. It should be borne in mind that measurement of only r phi and r psi distances for a decapeptide only defines the ( phi, psi)-space in terms of 4(10) possible conformations; the distances b-j served to reduce the degeneracy in possible (phi, psi)-space to one tyrocidine A conformation. The latter conformation is consistent with that derived from scalar coupling constants, hydrogen bonding studies, and proton-chromophore distance measurement, and closely resembles the conformation of gramicidin S.     

Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry

The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.     

A simple method for calculating the dissociation constant of a receptor (or enzyme).unlabeled ligand complex from radioligand displacement measurements

A general procedure is described for determining the dissociation constant of a receptor (or enzyme).unlabeled ligand complex (EI) by analyzing the I-dependent displacement of bound radioligand (A*) from EA*. The procedure (which involves measuring free A* in the presence of I) requires a knowledge of the total concentrations of receptor ([E]t), unlabeled ligand ([I]t) and radioligand ([A*]t), and the dissociation constant of the EA* complex. The unknown Kd is obtained from five simple, sequential calculations which are valid for either high or low affinity competitive unlabeled ligands and are independent of total receptor concentration or initial degree of saturation with A*. The procedure also provides the information needed to construct a distribution curve of all enzyme and ligand species (E, EA*, EI, A*, I) as [I]t is varied.