Effect of tryptic cleavage on the stability of myosin subfragment 1. Isolation and properties of the severed heavy-chain subunit

The procedure of thermal ion-exchange chromatography has been used to examine the effect of prior tryptic cleavage on the stability of myosin subfragment 1 (SF1). Although it is found that digestion does destabilize the subunit interactions at physiological temperatures, the heavy-chain subunit can be isolated either as an equimolar complex comprised of 50K, 27K, and 21K fragments or as one comprised of 50K, 27K, and 18K peptides. Thus, the interactions within the heavy chain are considerably more stable than those between the two subunits. Both forms of the free severed heavy chain exhibit ATPase properties similar to those of the parent tryptic SF1. The Vmax for the actin-activated MgATPase of the free severed heavy chain is the same as that for both undigested and tryptic SF1 (A2). Since its Km for actin is similar to that of tryptic SF1(A2), it may be concluded that changes in the affinity of SF1 for actin induced by trypsin [Botts, J., Muhlrad, A., Takashi, R., & Morales, M. F. (1982) Biochemistry 21, 6903-6905] are not dependent on the presence of the associated alkali light chain. Furthermore, the communication between the SH1 site and the ATPase site is also shown to be independent of the associated alkali light chain, and it persists despite the cleavages present in the free heavy chain. Studies on the ability of these severed heavy chains to reassociate with free A1 and A2 chains indicate that the binding site is retained in the 21K-severed heavy chain but is lost in the 18K form.     

Contents of myofibrillar proteins in cardiac, skeletal, and smooth muscles

The in situ contents of myosin, actin, alpha-actinin, tropomyosin, troponin, desmin were estimated in dog cardiac, rabbit skeletal, and chicken smooth muscles. Whole muscle tissues were dissolved with 8 M guanidine hydrochloride and subjected to two-dimensional gel electrophoresis, which is a nonequilibrium pH gradient electrophoresis (Murakami, U. & Uchida, K. (1984) J. Biochem. 95, 1577-1584) with some modification. The amount of protein in a spot on a slab gel was determined by quantification of the extracted dye. Dye binding capacity of individual myofibrillar proteins was determined by using the purified protein. Myosin contents were 82 +/- 7 pmol/mg wet weight in cardiac muscle, 105 +/- 10 pmol/mg wet weight in skeletal muscle, and 45 +/- 4 pmol/mg wet weight in smooth muscle. Actin contents were 339 +/- 15 pmol/mg wet weight in cardiac muscle, 625 +/- 27 pmol/mg wet weight in skeletal muscle, and 742 +/- 13 pmol/mg wet weight in smooth muscle. The subunit stoichiometry of myosin in the three types of muscles was two heavy chains and four light chains, and there was one light chain 2 for every heavy chain. The molar ratio of actin to tropomyosin was 7/1 in the three types of muscles. Striking differences were seen in the molar ratio of myosin to actin: 1.0/4.1 in cardiac muscle, 1.0/6.0 in skeletal muscle, and 1.0/16.5 in smooth muscle.     

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide-induced movements in the myosin head near the converter region

Structural changes in subfragment 1 of skeletal muscle myosin were investigated by cross-linking trypsin-cleaved S1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In the absence of nucleotide the alkali light chains are cross-linked to the 27 kDa heavy chain fragment; the presence of MgATP reduces the efficiency of this reaction. On the other hand, MgATP promotes the cross-link formation between the N-terminal 27 kDa and C-terminal 20 kDa fragments of the heavy chain. The chemical cleavage of the cross-linked heavy chains fragments with N-chlorosuccinimide and hydroxylamine indicates that the cross-links are formed between the regions spanning residues 131-204 and 699-809. These results indicate that the two regions of the heavy chain that are relatively distant in nucleotide-free skeletal S1 [Rayment et al. (1993) Science 261, 50-58] can potentially interact upon addition of nucleotide.     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

Purification of putative Ca2+ channel protein from rabbit skeletal muscle. Determination of the amino-terminal sequence

A putative Ca2+ channel protein was purified from rabbit skeletal muscle transverse tubules with the combined use of lectin affinity chromatography and ion-exchange chromatography, followed by sucrose density gradient centrifugation. The major component of the purified preparation detected by sodium dodecyl sulfate-gel electrophoresis was a protein of 150 kDa when reduced with 20 mM dithiothreitol and a 191-kDa protein when treated with 20 mM N-ethylmaleimide. Therefore, this protein appears to be identical with the alpha subunit previously described (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118). This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis, followed by electroelution and/or electroblotting, and its amino acid composition and NH2-terminal sequence were determined. The NH2-terminal sequence is: NH2-Glu-Pro-Phe-Pro-Ser-Ala-Val-X-Ile-Lys-Ser-X-Val-X-Lys-Met-Gln-.     

Amino acid sequence of the regulatory subunit of bovine type I adenosine cyclic 3',5'-phosphate dependent protein kinase

The complete amino acid sequence of the regulatory subunit of type I cAMP-dependent protein kinase from bovine skeletal muscle is presented. The S-carboxymethylated protein was cleaved with cyanogen bromide to provide a complete set of nonoverlapping fragments. These fragments were overlapped and aligned by using peptides generated by proteolytic cleavage. The protein contains 379 amino acid residues corresponding to a molecular weight of 42 804. As in the type II regulatory subunit of cAMP-dependent protein kinase, a pattern of internal gene duplication is observed, which is consistent with two cAMP-binding domains. The two types of regulatory subunit from type I and type II kinase display similarities in domain substructure and in amino acid sequence, which provide a molecular basis for new insight into their regulatory roles. Detailed analyses of the homology of the regulatory subunits of type I and type II cAMP-dependent protein kinase and of similar relationships to cGMP-dependent protein kinase and Escherichia coli catabolite gene activator protein are presented in accompanying reports from this laboratory [Takio, K., Smith, S. B., Krebs, E. G., Walsh, K., & Titani, K. (1984) Biochemistry (second paper of three in this issue); Takio, K., Wade, R. D., Smith, S. B., Krebs, E. G., Walsh, K. A., & Titani, K. (1984) Biochemistry (third paper of three in this issue)].     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Formation and properties of thermal hybrids

The formation of hybrid myosin and subfragment 1 species by incubation of these proteins with free alkali light chains at physiological ionic and temperature conditions is described. Exchange of bound alkali light chain on myosin by free alkali light chains under these conditions is readily demonstrated from the subunit composition of the isolated myosin. Therefore, the light chain exchange previously described for the one-headed subfragment 1 [Sivaramakrishnan, M., & Burke, M (1981) J. Biol. Chem. 256, 2607--2610] also occurs in the two-headed myosin molecule. It is found than the isozyme to hybrid transformation is dependent on both the temperature and the ionic strength of the incubation mixture but is relatively independent of pH in the range 6.5--8.0. A comparison of the SF1(A1) leads to SF1(A2)h system with the SF1(A2) leads to SF1(A1)h system indicates that more hybrid is formed in the latter case. With the assumption that hybrid formation reflects the degree of reversible dissociation exhibited by the isozyme, under the particular experimental condition employed, the data signify that the subunit interactions in the two isozymes are not identical and that the heavy chain--A1 interactions are significantly more stable that the heavy chain--A2 ones. An examination of the ATPase properties of the thermal hybrids in the presence and absence of actin indicates close similarities to their corresponding "native" isozymic counterparts.     

Effect of a single amino acid substitution on the folding of the alpha subunit of tryptophan synthase

The urea-induced unfolding of a missense mutant of the alpha subunit of tryptophan synthase from Escherichia coli involving the replacement of Gly by Glu at position 211 has been monitored by absorbance changes at 286 nm. Like the wild-type protein, the equilibrium unfolding curve demonstrates the presence of one or more stable intermediates. Comparison of these results with those from the wild-type alpha subunit [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] shows that the transition from the native conformation to the stable intermediates is displaced to higher urea concentration in the mutant alpha subunit; however, the transition from the intermediates to the unfolded form is unaffected. Kinetic studies show that the amino acid replacement slows the rate of unfolding by an order of magnitude. The effect on refolding rates is complex. One phase, previously assigned to proline isomerization [Crisanti, M. M., & Matthews, C. R. (1981) Biochemistry 20, 2700], is unaffected by the substitution. The rate of the second phase, which is urea dependent down to about 1 M urea, is slower than the corresponding phase in the wild-type protein by approximately a factor of 2. Below about 1 M urea, the rate of this phase becomes urea independent and identical with that of the wild-type alpha subunit. This change in urea dependence has been ascribed to a change in the nature of the rate-limiting step for this process from one involving folding to one involving proline isomerization. The results support the folding model for the alpha subunit proposed previously [Matthews, C. R., & Crisanti, M. M. (1981) Biochemistry 20, 784] and clarify the role of proline isomerization in limiting the rate of folding.     

Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase

The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural differences in the subfragment 1 and rod portions of myosin isozymes from adult and developing rat skeletal muscles

During development of fast contracting skeletal muscle in the rat hindleg, embryonic and neonatal forms of the myosin heavy chain are present prior to the accumulation of the adult fast type ( Whalen , R. G., Sell, S. M., Butler-Browne, G.S., Schwartz, K., Bouveret, P., and Pinset -H arstr ?m, I. (1981) Nature (Lond.) 292, 805-809). Polypeptide mapping of the heavy chain subunit using partial proteolysis in the presence of sodium dodecyl sulfate has shown differences in the cleavage patterns for these various heavy chains. Using this technique, we have now examined subfragments, which represent functional domains, from several different myosin isozymes. The heavy chains of the S-1 subfragments containing either light chain 1 or light chain 3 are indistinguishable for the neonatal or fast myosin isozymes. We also isolated the S-1 fragments and the alpha-helical COOH-terminal half of the molecule (rod) from rat embryonic, neonatal, and adult fast and slow myosin, as well as myosin from cardiac ventricles. All of these S-1 and rod fragments were different, indicating that the previously reported differences among these different myosin heavy chain isozymes are located in both the S-1 and rod subfragments for all myosins examined. However, the polypeptide maps of neonatal and adult fast S-1 show clear similarities, as do the maps of slow and cardiac S-1. These similarities in the two pairs of polypeptide maps were confirmed by the results of immunoblotting experiments using antibodies to adult fast and to slow myosin.     

Isolation of heavy chain isoenzymes of myosin subfragment 1 by high performance ion exchange chromatography

The procedure of high performance ion-exchange chromatography has been used to fractionate subfragment 1 of myosin (SF1) into its isoenzymic forms. In contrast to conventional ion-exchange procedures which yield two fractions corresponding to SF1(A1) and SF1(A2), the high performance liquid chromatography (HPLC) procedure resolves SF1 into four discrete fractions. The first pair that is eluted appears to be A1-containing isoenzymes while the latter pair corresponds to the A2 forms based on their polypeptide compositions by gel electrophoresis in the presence of sodium dodecyl sulfate. By gel electrophoresis under nondenaturing conditions it is not possible to differentiate between the two fractions corresponding to each isoenzyme. Although very minor differences between the fractions can be seen by the presence of extraneous peptides, these are present in far below stoichiometric amounts and, therefore, make it very unlikely that the superior fractionation by the HPLC procedure is based on their presence. An examination of the heavy chain heterogeneity in each of these fractions by peptide mapping revealed that the extra separation was based on this factor. Thus the HPLC procedure is capable of providing separation of SF1 into heavy chain-based isozymes as well as the light chain forms. ATPase measurements of these fractions reveal only minor differences in the Ca2+- and EDTA-activated ATPase.     

Evidence for the association between two myosin heads in rigor acto-smooth muscle heavy meromyosin

The rigor complexes that formed between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), in which the heavy chains had been cleaved with trypsin into 24K, 50K, and 68K fragments, were examined by using the zero-length chemical cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Two cross-linked products of approximate Mr 115K and 60K were generated. These products were not obtained by EDC treatment of HMM in the absence of F-actin. The HMM fragments that participated in cross-linking were identified by fluorescent labeling and amino acid composition studies. The 115K peptide was determined to be a covalently cross-linked complex that formed between actin and the COOH-terminal 68K fragment of the HMM heavy chain. Our results are in agreement with a previous study which proposed that the site of cross-linking between HMM and F-actin resides within the COOH-terminal 22K fragment of the myosin subfragment 1 heavy chain [Marianne-Pépin, T., Mornet, D., Bertrand, R., Labbé, J.-P., & Kassab, R. (1985) Biochemistry 24, 3024-3029]. The 60K peptide, however, was not a product of cross-linking between HMM and F-actin. On the basis of its amino acid composition, we concluded that this 60K peptide was a cross-linked dimer of the NH2-terminal 24K fragments of the HMM heavy chain. The cross-linking of acto-gizzard HMM significantly increased the Mg-ATPase activity of gizzard HMM without any observable phosphorylation of the regulatory (20K) light chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Location of an essential carboxyl group along the heavy chain of cardiac and skeletal myosin subfragments 1

Cardiac and skeletal myosin subfragments 1 cleaved into three fragments were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene-sulfonate in the presence of the nucleophile nitrotyrosine ethyl ester. The effects observed (first-order kinetics of ATPase inactivation, incorporation of 1 mol of nitrotyrosine/mol of subfragment 1) were similar to those previously observed for the nondigested subfragments 1 [Lacombe, G., Van Thiem, N., & Swynghedauw, B. (1981) Biochemistry 20, 3648-3653; K?rner, M., Van Thiem, N., Lacombe, G., & Swynghedauw, B. (1982) Biochem. Biophys. Res. Commun. 105, 1198-1207]. For both native and digested subfragments 1, which were inactivated to the extent of about 70%, the location of the label nitrotyrosine was performed by immunological blotting with 125I-labeled anti-nitrotyrosine immunoglobulins. It was found that the modified residue was essentially located on the heavy chain for the native subfragments 1 and on the 50K peptide for the digested subfragments 1.     

Identification of aspartate-184 as an essential residue in the catalytic subunit of cAMP-dependent protein kinase

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) was previously shown to be an irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase, and MgATP protected against inactivation [Toner-Webb, J., & Taylor, S. S. (1987) Biochemistry 26, 7371]. This inhibition by DCCD indicated that an essential carboxyl group was present at the active site of the enzyme even though identification of that carboxyl group was not possible. This presumably was because a nucleophile on the protein cross-linked to the electrophilic intermediate formed when the carbodiimide reacted with the carboxyl group. To circumvent this problem, the catalytic subunit first was treated with acetic anhydride to block accessible lysine residues, thus preventing intramolecular cross-linking. The DCCD reaction then was carried out in the presence of [14C]glycine ethyl ester in order to trap any electrophilic intermediates that were generated by DCCD. The modified protein was treated with trypsin, and the resulting peptides were separated by HPLC. Two major radioactive peptides were isolated as well as one minor peptide. MgATP protected all three peptides from covalent modification. The two major peaks contained the same modified carboxyl group, which corresponded to Asp-184. The minor peak contained a modified glutamic acid, Glu-91. Both of these acidic residues are conserved in all protein kinases, which is consistent with their playing essential roles. The positions of Asp-184 and Glu-91 have been correlated with the overall domain structure of the molecule. Asp-184 may participate as a general base catalyst at the active site. A third carboxyl group, Glu-230, also was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbodiimide-catalyzed cross-linking sites in the heads of gizzard heavy meromyosin attached to F-actin

In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature

Evidence is presented that under physiological conditions of ionic strength and temperature, where myosin Subfragment 1 is hydrolyzing MgATP, the interaction between its subunits is extremely labile. Incubation of [3H]N-ethylmaleimide-SF1(A1) with N-ethylmaleimide-SF1(A2) in the presence of 10 mM MgATP at 37 degrees C resulted in the exchange of subunits between these isozymes. This is readily discernible from the subunit composition and distribution of the 3H label after separation of the isozymes by ion exchange chromatography. Moreover, incubation of unmodified SF1(A1) or SF1(A2) with the free Alkali light chains A2 and A1, respectively, under the same conditions led to the formation of significant amounts of the hybrid species. These findings suggest that in vivo the Alkali light chain-heavy chain interaction of Subfragment 1 is in a state of dynamic equilibrium between associated and dissociated states.     

A myofibrillar protein phosphatase from rabbit skeletal muscle contains the beta isoform of protein phosphatase-1 complexed to a regulatory subunit which greatly enhances the dephosphorylation of myosin.

A form of protein phosphatase-1 (PP1M), which possesses 25-fold higher activity towards the P light chain of myosin (in heavy meromyosin) than other forms of protein phosphatase-1, was purified over 200,000-fold from the myofibrillar fraction of rabbit skeletal muscle. PP1M, which eluted from Superose 12 with an apparent molecular mass of 60 kDa, was dissociated by LiBr into two subunits. One of these displayed enzymic properties identical to those of the catalytic subunit of protein phosphatase-1 (PP1C) and was identified as the beta isoform of PP1C by amino acid sequencing. The second subunit had no intrinsic protein phosphatase activity, but greatly increased the rate at which PP1C dephosphorylated skeletal-muscle heavy meromyosin and decreased the rate at which it dephosphorylated glycogen phosphorylase. The properties of PP1M, together with those of smooth muscle PP1M [Alessi, D., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035] and the previously characterised glycogen-associated form of protein phosphatase-1 (PP1G), indicate that the subcellular localisation and substrate specificity of PP1 is determined by its interaction with specific targetting subunits.     

Nuclear overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (chi = 78 +/- 10 degrees) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations of MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH3)4ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides [chi = 78 +/- 10 degrees, O1'-endo; Rosevear, P.R., Bramson, H.N., O'Brian, C., Kaiser, E.T., & Mildvan, A.S. (1983) Biochemistry 22, 3439]. Distance geometry calculations also suggest that upper limit distances, when low enough (less than or equal to 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker.     

Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese.

In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins. The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese. These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P. V., Lubben, T. H., Reed, J., Goloubinoff, P., O'Keefe, D. P., and Lorimer, G. H. (1990) Biochemistry 29, 5665-5671). The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60. The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10. Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP. At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins. This spontaneous refolding can be arrested by chaperonin 60. There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP. ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins. Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). The chaperonin proteins appear to play a similar role in as much as they can replace the detergents. Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60. The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added. The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.