Závodszky, P., Kardos, J., Svingor & Petsko, G. A.

Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins.

Proc Natl Acad Sci U S A 95, 7406-11.

3-Isopropylmalate dehydrogenase (IPMDH, E.C. from the thermophilic bacterium Thermus thermophilus HB8 is homologous to IPMDH from the mesophilic Escherichia coli, but has an approximately 17 degreesC higher melting temperature. Its temperature optimum is 22-25 degreesC higher than that of the E. coli enzyme; however, it is hardly active at room temperature. The increased conformational rigidity required to stabilize the thermophilic enzyme against heat denaturation might explain its different temperature-activity profile. Hydrogen/deuterium exchange studies were performed on this thermophilic-mesophilic enzyme pair to compare their conformational flexibilities. It was found that Th. thermophilus IPMDH is significantly more rigid at room temperature than E. coli IPMDH, whereas the enzymes have nearly identical flexibilities under their respective optimal working conditions, suggesting that evolutionary adaptation tends to maintain a "corresponding state" regarding conformational flexibility. These observations confirm that conformational fluctuations necessary for catalytic function are restricted at room temperature in the thermophilic enzyme, suggesting a close relationship between conformational flexibility and enzyme function.

Várallyay, E., Pál, G., Patthy, A., Szilágyi, L. & Gráf, L.

Two mutations in rat trypsin confer resistance against autolysis.

Biochem Biophys Res Commun 243, 56-60.

Due to autodigestion the activity of dissolved trypsin successively decreases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysis sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val118 and Lys145-Ser146. Out of these three sites only the first two exist in rat trypsin, an enzyme that has been the target of protein engineering for more than ten years. In this work Lys61 and Arg117 were replaced by Asn via site directed mutagenesis to transform the corresponding peptide bonds to trypsin resistant ones. Kinetic parameters of K61N, R117N and the double mutant K61N/R117N are practically identical with those of the wild-type enzyme. By contrast, the rate of autolysis of each singly-substituted species is substantially slower than with the parent trypsin. In particular, the double mutant shows dramatically increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequence determination. We propose a model to explain why these two positions play a key role in autolysis and how Ca2+ can influence this process. In addition, our in vitro results strongly support the recently proposed model of human hereditary pancreatitis.

Málnási-Csizmadia, A., Shimony, E., Hegyi, G., Szent-Györgyi, A. G. & Nyitray, L.

Dimerization of the head-rod junction of scallop myosin.

Biochem Biophys Res Commun 252, 595-601.

We have compared the dimerization properties and coiled-coil stability of various recombinant fragments of scallop myosin around the head-rod junction. The heavy-chain peptide of the regulatory domain and its various extensions toward the alpha-helical rod region were expressed in Escherichia coli, purified, and reconstituted with the light chains. Rod fragments of the same length but without the light-chain binding domain were also expressed. Electron micrographs show that the regulatory domain complex containing 340 residues of the rod forms dimers with two knobs (two regulatory domains) at one end attached to an approximately 50-nm coiled coil. These parallel dimers are in equilibrium with monomers (Kd = 10.6 microM). By contrast, complexes with shorter rod extensions remain predominantly monomeric. Dimers are present, accounting for ca. 5% of the molecules containing a rod fragment of 87 residues and ca. 30% of those with a 180-residue peptide. These dimers appear to be antiparallel coiled coils, as judged by their length and the knobs observed at the two ends. The rod fragments alone do not dimerize and form a coiled-coil structure unless covalently linked by disulfide bridges. Our results suggest that the N-terminal end of the coiled-coil rod is stabilized by interactions with the regulatory domain, most likely with residues of the regulatory light chain. This labile nature of the coiled coil at the head-rod junction might be a structural prerequisite for regulation of scallop myosin by Ca2+-ions.

Lengyel, Z., Pál, G. & Sahin-Tóth, M.

Affinity purification of recombinant trypsinogen using immobilized ecotin.

Protein Expr Purif 12, 291-4.

Affinity purification of inactive precursors (zymogens) of serine proteases on protease inhibitor columns is not feasible, due to the weak interaction between canonical protease inhibitors and protease zymogens. In this study we demonstrate that immobilized ecotin, a unique protease inhibitor from Escherichia coli, provides a superior affinity matrix for the purification of trypsinogen and possibly other serine protease zymogens as well.

Kim, E., Phillips, M., Hegyi, G., Mühlrad, A. & Reisler, E.

Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers.

Biochemistry 37, 17793-800.

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.

Kim, E., Bobkova, E., Miller, C. J., Orlova, A., Hegyi, G., Egelman, E. H., Mühlrad, A. & Reisler, E.

Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin.

Biochemistry 37, 17801-9.

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

Hegyi, G., Mak, M., Kim, E., Elzinga, M., Mühlrad, A. & Reisler, E

Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

Biochemistry 37, 17784-92.

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.