α-Helix-to-random-coil transitions of two-chain coiled coils: Experiments on the thermal denaturation of ββ tropomyosin cross-linked selectively at C-190

A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO₄/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics.     

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)     

The kinetics of chain exchange in two-chain coiled coils: alpha alpha- and beta beta-tropomyosin.

Measurements are presented on the time course of chain exchange among two-chain alpha-helical coiled coils of rabbit tropomyosin. All experiments are in a regime (temperature, protein concentration) in which coiled-coil dimers are the predominant species. Self-exchange in alpha alpha-tropomyosin was investigated by mixing alpha alpha species with alpha* alpha*, the asterisk designating an alpha-chain whose lone sulfhydryl (C190) has been blocked by carboxyamidomethylation. The overall process alpha alpha + alpha* alpha* in equilibrium with 2 alpha alpha* is followed by measurement of the fraction (h) of alpha alpha* species as a function of time. Similarly, self-exchange in beta beta-tropomyosin is examined by measurements of the overall process: beta beta + beta* beta* in equilibrium with 2 beta beta*, in which beta* signifies a beta-chain blocked at both sulfhydryls (C36 and C190). The observed time course for both chains is well fit by the first-order equation: h (t) = h (infinity) (1-e-k1t), with h (infinity) congruent to 0.5. This long-time limit is as expected for self-exchange, and agrees with experiments that attain equilibrium after slow cooling of thermally dissociated and unfolded chains. The simplest consonant mechanism is chain exchange by rate-limiting dissociation of dimers followed by random reassociation. Kinetic analysis shows k1 to be the rate constant for the chain dissociation step, a quantity not previously measured for any coiled coil. This rate constant for beta beta species is about an order of magnitude greater than for alpha alpha. In both, the activation enthalpy and entropy are very large, suggesting that activation to an extensively (greater than 50%) unfolded species necessarily precedes dissociation. Experiments are also reported for overall processes: alpha alpha + beta* beta* in equilibrium with 2 alpha beta* and alpha* alpha* + beta beta in equilibrium with 2 alpha* beta. Results are independent of which chain is blocked. Again h (infinity) congruent to 0.5, in agreement with equilibrium experiments, and the time course is first order. The rate constants and activation parameters are intermediate between those for self-exchange.     

Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin

The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha.     

Beta beta homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation.

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 3-4/1). In some extant studies, no beta beta molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of beta beta molecules cross-linked at both C36 and C190 (beta = beta). In such cross-linked RSTm, 3-8% beta = beta is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes approximately 4-10% beta beta when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed beta beta species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55 degrees C or by incubating at 45 degrees C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of beta beta increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).     

Thermal unfolding equilibria in homodimeric chicken gizzard tropomyosin coiled coils

CD studies are presented on thermal unfolding of coiled-coil homodimers of two genetic variant chains of chicken gizzard tropomyosin (CG-Tm). The experiments include the effects of cross-linking both isoforms and the dependence on protein concentration of unfolding in both reduced isoforms, variables not examined in extant work. The general shapes of the unfolding curves for singly cross-linked species depend on whether the crosslink is at C190 (its site on one isoform) or at C36 (its site on the other). These curves are compared with extant ones for various cross-linked species of rabbit tropomyosin. The comparison supports the view that the unfolding behavior of cross-linked species results from a complex interaction of strain at the cross-link, local variations in structural stability, and loop entropy. The observed concentration dependence of the transition temperature for the uncross-linked (reduced) species of CG-Tm is very small (2.9°C) for one variant homodimer and unobservably small (< 2°C) for the other in the 100-fold concentration range (～ 0.01–1.0 mg/mL) accessible here. These experimental values of ΔT_m are much smaller than are predicted from extant values of the van't Hoff transition enthalpies, calling the latter into question. © 1994 John Wiley & Sons, Inc.     

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.     

Chemical cross-linking of Ia alloantigen alpha and beta chains with dimethyl 3,3'-dithiobispropionimidate.

We have examined the polypeptide chain composition of membrane-bound and detergent-solubilized Ia antigens using the chemical cross-linking reagent dimethyl 3,3'-dithiobispropionimidate (DTBP). Products of the I-E/C subregion of the major histocompatibility complex, which were solubilized from spleen cells with the detergent NP-40 and partially purified by affinity chromatography on lentil lectin-agarose, could be almost completely cross-linked by DTBP. Thus, the characteristic 33,000 m.v. (alpha) and 28,000 (beta) polypeptide chains seen on sodium dodecylsulfate polyacrylamide gels disappeared and a major new species of 60,000 m.w. appeared after cross-linking. When isolated and reduced with 2-mercaptoethanol, the 60,000 m.w. peak was found to be comprised to alpha and beta chains. Similar results were obtained when I-E/C, as well as I-A, alpha and beta chains were crosslinked on the cell surface. These data demonstrate that the alpha and beta chains of the Ia antigens exist primarily in the form of a dimer both in detergent solution and in situ.     

Thermally induced chain exchange of frog alpha beta-tropomyosin.

The thermally induced unfolding of the alpha-helix of Rana esculenta alpha alpha, alpha beta and beta beta tropomyosin and two tryptic fragments approximately corresponding to the N- and C-terminal halves of alpha beta have been investigated by use of optical rotation, circular dichroism and UV difference spectroscopy. Reversible unfolding transitions of alpha alpha and beta beta occur around 49 degrees C and 32 degrees C, respectively. The helix unfolding of alpha beta shows two major transitions at 36 degrees C and 48 degrees C, with only the latter being reversible. The major unfolding transitions of each of the N- and C-terminal alpha beta peptides roughly correspond to the low and high temperature transitions of intact alpha beta, respectively. This suggests that the unfolding of alpha beta could be due to unfolding of two independent domains in alpha beta. UV difference data, crosslinking and chromatography results show, however, that the unfolding of alpha beta at 36 degrees C is due to chain exchange with the formation of alpha alpha homodimers and largely unfolded beta monomers, and that the transition at 48 degrees C is due to unfolding of alpha alpha dimers.     

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins.

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.     

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.     

Primary structure of alpha and beta chains from the major hemoglobin component of the magpie goose (Anseranas semipalmata, Anatidae)

The amino acid sequence of the alpha and beta chains from the major hemoglobin component (HbA) of Australian Magpie Goose (Anseranas semipalmata) is given. The minor component with the alpha D chains was detected, but only found in low concentrations. By homologous comparison, Greylag Goose hemoglobin (Anser anser) and Australian Magpie Goose alpha chains differ by 13 amino acids or 17 nucleotide (4 two point mutations) exchanges, beta chains by 6 exchanges. Seven alpha 1 beta 1 contacts are modified by substitutions in positions alpha 30-(B11)Glu leads to Gln, alpha 34(B15)Thr leads to Gln, alpha 35(B16)-Ala leads to Thr, alpha 36(B17)Tyr leads to Phe, beta 55(D6)Leu leads to Ile, beta 119(GH2)Ala leads to Ser and beta 125(H3)Glu leads to Asp. Further, one alpha 1 beta 2 contact point was changed in beta 39(C5)Gln leads to Glu. Mutation in this position, except in two abnormal human hemoglobins, was not found in any species. Amino acid exchanges between hemoglobin of Australian Magpie Goose and other birds are discussed.     

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.     

Reaction of haptoglobin with hemoglobin covalently cross-linked between the alpha beta dimers.

Hemoglobin tetramers which cannot split into alphabeta dimers, because they are covalently cross-linked between the beta chains across the polyphosphate binding site, form complexes with haptoglobin. The reaction is biphasic as measured by fluorescence quenching and peroxidase activity. A complex in which one of the alpha beta dimers of the cross-linked hemoglobin is bound to one of the sites in the divalent haptoglobin molecule, is formed reversibly during the initial fast phase. In the subsequent slower step, this product then either polymerizes, adds another cross-linked hemoglobin molecule or, in the presence of excess haptoglobin, combines with a second haptoglobin molecule. This latter complex, in which two haptoglobin molecules are bridged by a cross-linked hemoglobin tetramer, can still combine with normal alpha beta dimers at the vacant haptoglobin combining sites. In spite of the very low oxygen affinity of the cross-linked hemoglobin, combination with haptoglobin shifts if oxygen affinity to the very high value of the normal hemoglobin-haptoglobin complex.     

Double helices with parallel strands are formed by nuclease-resistant oligo-[alpha]-deoxynucleotides and oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent with complementary oligo-[beta]-deoxynucleotides

Oligo-[alpha]-thymidylates have been synthesized and covalently linked to an intercalating agent (an acridine derivative) and/or to a p-azidophenacyl group. These molecules bind to a complementary oligo-[beta]-deoxynucleotide. A strong stabilization is obtained by covalent attachment of the acridine derivative at the 5' end of the oligo-[alpha]-deoxynucleotide. Upon excitation of the p-azidophenacyl group with ultraviolet light, the oligo-[alpha]-thymidylate is crosslinked to its target sequence. These crosslinks are converted to chain breaks under alkaline conditions. This allows an unambiguous assignment of the orientation of the two oligonucleotide chains. As expected, beta-beta hybrids have an antiparallel orientation, whereas the two chains of alpha-beta hybrids are parallel independently of whether an intercalating agent is covalently linked to the alpha-oligo-nucleotide. Oligo-[alpha]-thymidylates covalently linked to an acridine derivative are highly resistant to endo- and exonucleases. Therefore, they could be used as anti-messengers to block mRNA translation in vivo under conditions where oligo-[beta]-deoxynucleotides are usually hydrolysed.     

Cross-linking of two beta subunits in the closed conformation in F1-ATPase

In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP.     

The (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 has altered ATPase activity after cross-linking alpha and beta subunits at noncatalytic site interfaces

In crystal structures of bovine MF(1), the side chains of alpha F(357) and beta R(372) are near the adenines of nucleotides bound to noncatalytic sites. To determine if during catalysis these side chains must pass through the different arrangements in which they are present in crystal structures, the catalytic properties of the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex of the TF(1)-ATPase were characterized before and after cross-linking the introduced cysteines with CuCl(2). The unmodified mutant enzyme hydrolyzes MgATP at 50% the rate exhibited by wild type. Detailed comparison of the catalytic properties of the double mutant enzyme before and after cross-linking with those of the wild-type subcomplex revealed the following. Before cross-linking, the (alpha F(357)C)(3)(beta R(372)C)(3)gamma subcomplex has less tendency than wild type to release inhibitory MgADP entrapped in a catalytic site during turnover when MgATP binds to noncatalytic sites. Following cross-linking, ATPase activity is reduced 5-fold, and inhibitory MgADP entrapped in a catalytic site during turnover does not release under conditions wherein binding of ATP to noncatalytic sites of the wild-type enzyme promotes release of MgADP from the affected catalytic site. When assayed in the presence of lauryldimethylamine oxide, which prevents turnover-dependent entrapment of inhibitory MgADP in a catalytic site, ATPase activity of the cross-linked form is 47% that of the unmodified mutant enzyme. These results suggest that, during catalysis, the side chains of alpha F(357) and beta R(372) do not pass through the extremely different relative positions in which they exist at the three noncatalytic site interfaces in crystal structures.     

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).     

Preferential assembly of the tropomyosin heterodimer: equilibrium studies

Thermal unfolding/refolding studies of the three tropomyosin dimers, alpha alpha, alpha beta, and beta beta, from chicken gizzard muscle were performed to explain the preferential assembly of alpha- and beta-tropomyosin subunits into heterodimers, alpha beta [Lehrer, S. S., & Qian, Y. (1989) J. Biol. Chem. 265, 1134]. Circular dichroism measurements showed that all three dimers unfolded in cooperative reversible transitions with T1/2 = 40.0 degrees C and delta H degrees = 162 kcal/mol for alpha alpha and with T1/2 = 42.6 degrees C and delta H degree = 98 kcal/mol for beta beta at 0.4-0.5 microM concentrations. Fluorescence measurements on pyrenyliodoacetamide-labeled tropomyosin showed that (i) excimer fluorescence decreases in parallel with unfolding of homodimers, (ii) at physiological temperature, heterodimers are formed from micromolar mixtures of homodimers over a period of minutes, and (iii) heterodimers unfold/refold with temperature without appreciable formation of homodimers. To understand the preferential formation of alpha beta, we calculated the concentrations of all species present as a function of temperature for equal total amounts of alpha and beta, using the measured thermodynamic constants of the unfolding/dissociation equilibria for alpha alpha and beta beta. Values for delta H degrees = 225 kcal/mol and T1/2 = 43 degrees C for unfolding of alpha beta at 0.5 microM concentration were obtained from the best fit of the calculations to the measured helical content vs temperature of alpha beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.