Ydj1p二聚体中β14~β15与domain-III分离的拉伸分子动力学模拟研究

分子伴侣Hsp40是一种以二聚体的形式调控非天然多肽折叠的热激蛋白。本文通过拉伸分子动力学研究了酵母Hsp40家族成员Ydj1p二聚体中β14-β15与domain-Ⅲ的分离过程,深入探讨了影响Ydj1p二聚体稳定性的重要残基和相互作用力。研究表明,残基Thr366、Asp368、Cys370、Leu372和Phe375在Ydj1P二聚体的形成过程中发挥着重要的作用。其中,β14-β15中的残基Thr366和Asp368分别通过与domain-Ⅲ内的残基Asp291、Trp292和Trp292、Lys294之间形成的氢键,Asp368通过与domain-Ⅲ内的残基Lys314形成盐桥,Cys370、Leu372和Phe375则是通过与domain-Ⅲ形成疏水作用力来稳定Ydj1p二聚体结构。     

Improved method for mapping the binding site of an actin-binding protein in the actin sequence. Use of a site-directed antibody against the N-terminal region of actin as a probe of its N-terminus

An antibody was raised against the N-terminal 18 residues of rabbit skeletal muscle actin. By the use of this antibody as the N-terminal probe of actin and the fluorescent label at Cys-374 as its C-terminal probe, binding sites of depactin (an actin-depolymerizing protein from starfish oocytes) were identified in the actin sequence according to the method of Sutoh [Sutoh, K. (1982) Biochemistry 21, 3654-3661]. Cross-linking of the one-to-one complex of actin and depactin with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC) generated two types of cross-linked products with slightly different apparent molecular weights, denoted as 60KU and 60KL. By the use of the N-terminal probe, it was unequivocally revealed that the C-terminal actin segment of residues 357-375 participated in cross-linking with depactin to form 60KL. On the other hand, by the use of the C-terminal probe it was revealed that the N-terminal actin segment of residues 1-12 participated in cross-linking with depactin to form 60KU. Since EDC cross-links Lys residue with Asp or Glu residue only when they are in direct contact, the result indicates that some of the N-terminal residues 1-12 and the C-terminal residues 357-375 of actin participate in binding depactin. The introduction of the N-terminal probe (the antibody recognizing the actin N-terminus) has increased the flexibility of the mapping method for locating binding sites of actin-binding proteins in the actin sequence.     

Probing Dictyostelium severin structure and function by cross linking to actin.

DS151 is the first 151 amino acids of the Dictyostelium discoidium protein severin, which shares high sequence similarity with segment 1 of the actin-severing protein gelsolin. DS151 is able to mediate the depolymerization of F-actin in a calcium-dependent fashion, much like segment 1 of gelsolin. A structural model of DS151 was obtained by comparative modeling studies with segment 1 of gelsolin. This model was tested by studies of chemical cross linking between DS151 and bound actin, suggesting that Cys residues on DS151 are cross linked with Lys residues of actin. The model suggests that Cys125 of DS151 cross links with either Lys326 or Lys328 of actin. Mutagenesis of DS151 demonstrates that Cys125 of DS151 dominates the cross linking, whereas Cys25 of DS151 makes a minor contribution through a longer-range cross link with Cys374 of actin, which likely involves flexibility of both proteins in that region.     

Relocation of Cys374 of actin induced by labeling with fluorescent dyes

Cys374 is an important landmark of actin, because its sulfhydryl group is reactive and can be labeled with various reagents. The atomic coordinates of actin Cys374 have been determined by X-ray crystallography of co-crystals of actin with either profilin or gelsolin. However, the positions of Cys374 in the crystals determined were not consistent with the data obtained through fluorescence resonance energy transfer. Here, we examined its position by means of probabilistic distance geometry using published fluorescence resonance transfer data and found that Cys374 of actin was relocated when Cys374 was labeled with fluorescent dyes that caused steric hindrance. The atomic coordinates of Cys374 after heavy atom labeling have been found to be different from those for the native crystal. This is consistent with our results. Therefore, the position of Cys374 is sensitive to chemical modification that introduces a bulky reagent. This also suggests that the conformation of the C-terminal region of actin could also be sensitive to the environment.     

Unidirectional movement of an actin filament taking advantage of temperature gradients

An actin filament with heat acceptors attached to its Cys374 residue in each actin monomer could move unidirectionally even under heat pulsation alone, while in the total absence of both ATP and myosin. The prime driver for the movement was temperature gradients operating between locally heated portions on an actin filament and its cooler surroundings. In this report, we investigated how the mitigation of the temperature gradients induces a unidirectional movement of an actin filament. We then observed the transversal fluctuations of the filament in response to heat pulsation and their transition into longitudinally unidirectional movement. The transition was significantly accelerated when Cys374 and Lys336 were simultaneously excited within an actin monomer. These results suggest that the mitigation of the temperature gradients within each actin monomer first went through the energy transformation to transversal fluctuations of the filament, and then followed by the transformation further down to longitudinal movements of the filament. The faster mitigation of temperature gradients within actin monomer helps build up the transition from the transversal to longitudinal movements of the filament by coordinating the interaction between the neighboring monomers.     

Biosynthesis of bacterial glycogen. Determination of the amino acid changes that alter the regulatory properties of a mutant Escherichia coli ADP-glucose synthetase

The ADP-glucose synthetase of Escherichia coli K12 mutant 618 has a higher apparent affinity for the activator, fructose 1,6-P2 and a lower apparent affinity for the inhibitor, 5'-AMP, than the normal enzyme. The structural gene, glgC, of the mutant enzyme has been cloned and sequenced (Lee, Y. M., Kumar, A., and Preiss, J. (1987) Nucleic Acids Res. 15, 10603). Substitutions in the mutant enzyme were amino acid residues 296 (Lys to Glu) and 336 (Gly to Asp). Single mutant enzymes, Glu296 and Asp336, were constructed using oligonucleotide-directed mutagenesis. The Glu296 enzyme had the same allosteric kinetic constants as the wild type enzyme. The Asp336 enzyme was catalytically defective. Thus, the mutations at 296 and at 336 separately could not account for the allosteric alterations of the mutant enzyme. A hybrid glgC gene was prepared from genes of wild type and mutant 618 glgC using DNA recombinant techniques. The C-terminal portion of mutant 618 containing Glu296 and Asp336, combined with the N-terminal portion of wild type enzyme, showed allosteric and substrate kinetics similar to mutant 618 enzyme. Thus, alteration of the normal allosteric properties in mutant 618 are due to changes of both Lys296 to Glu and Gly336 to Asp.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Removing the two C-terminal residues of actin affects the filament structure.

We define conditions under which the two C-terminal residues of actin, Cys-374 and Phe-375, can be selectively removed by proteolysis with trypsin. This modification had little effect on the secondary structure of actin detected by Fourier-transform infrared spectroscopy. However, removing these residues caused small but significant decreases in the critical concentration of actin, in its ability to activate myosin ATPase, and in its interaction with tropomyosin and troponin. Removing residues 374-375 caused dramatic changes in the actin filament as seen by electron microscopy. The filaments had a much greater and more irregular curvature and were intertwined into disordered multifilament bundles. Removing 374-375 also significantly lowered the flow viscosity of filamentous-actin solutions. These data suggest an increase in the flexibility and fragility of the filament, supporting the idea that the C-terminus forms one of the major intermonomer contacts in the filament.     

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.     

One plant actin isovariant, ACT7, is induced by auxin and required for normal callus formation.

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.     

HU-1 mutants of Escherichia coli deficient in DNA binding

We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.     

Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.     

Flexibility of myosin-subfragment-1 in its complex with actin as revealed by fluorescence resonance energy transfer.

The flexibility of the acto-myosin complex in rigor conditions was characterized by measuring the temperature profile of normalized fluorescence resonance energy transfer efficiency, f' [Somogyi, B., Matkó, J., Papp, S., Hevessy, J., Welch, G.R. & Damjanovich, S. (1984) Biochemistry 23, 3403-3411]. Fluorescence acceptors were introduced to the Cys374 residues of actin and the donors were covalently attached either to Cys707 in the catalytic domain or to Cys177 in the essential light-chain of myosin S1. Fluorescence resonance energy transfer measurements revealed that the protein matrix between Cys374 of actin and Cys707 of S1 is rigid. In contrast, the link between the catalytic and light-chain-binding domains in myosin S1 is flexible. We have recently shown that the positional distribution of Cys707 was narrow relative to the actin filament, while that of the Cys177 was broad. Accordingly, the broad positional distribution of Cys177 is likely to be due to the large flexibility of the link between the catalytic and light-chain-binding domains. This flexibility is probably essential for the interdomain reorganization of the myosin head during the force generation process and for accommodating the symmetry difference between actin and myosin filaments to allow the formation of cross-bridges.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.     

The effect of the dilated cardiomyopathy-causing Glu40Lys TPM1 mutation on actin-myosin interactions during the ATPase cycle

Dilated cardiomyopathy (DCM), characterized by cardiac dilatation and contractile dysfunction, is a major cause of heart failure. DCM can result from mutations in the gene encoding cardiac α-tropomyosin (TM). In order to understand how the dilated cardiomyopathy-causing Glu40Lys mutation in TM affects actomyosin interactions, thin filaments have been reconstituted in muscle ghost fibers by incorporation of labeled Cys707 of myosin subfragment-1 and Cys374 of actin with fluorescent probe 1.5-IAEDANS and α-tropomyosin (wild-type or Glu40Lys mutant). For the first time, the effect of these α-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. The Glu40Lys mutant TM inhibited these movements at the transition from AM^∗∗·ADP·Pi to AM state, indicating a decrease of the proportion of the strong-binding sub-states in the actomyosin population. These structural changes are likely to underlie the contractile deficit observed in human dilated cardiomyopathy.     

Old yellow enzyme protects the actin cytoskeleton from oxidative stress

Old Yellow Enzyme (OYE) has long served as a paradigm for the study of flavin-containing NADPH oxido-reductases and yet its physiological role has remained a mystery. A two-hybrid interaction between Oye2p and actin led us to investigate a possible function in the actin cytoskeleton. We found that oye deletion strains have an overly elaborate actin cytoskeleton that cannot be attributed to changes in actin concentration but likely reflect stabilization of actin filaments, resulting in excessive actin assembly. Cells expressing the actin mutant act1-123p, which has a weakened interaction with Oye2p, show comparable defects in actin organization to the oye deletion strain that can be suppressed by overexpression of Oye2p. Similarly, mutation of either conserved cysteine of the potential disulfide pair Cys285-Cys374 in actin completely suppresses the actin organization defect of the oyeDelta phenotype. Strains lacking Oye function are also sensitive to oxidative stress as induced by H2O2, menadione, and diamide treatment. Mutation of either Cys285 or Cys374 of actin suppresses the sensitivity of oyeDelta strains to oxidative stress and in fact confers super-resistance to oxidative stress in otherwise wild-type strains. These results suggest that oxidative damage to actin, like that which has been observed in irreversibly sickled red blood cells, may be a general phenomenon and that OYE functions to control the redox state of actin thereby maintaining the proper plasticity of the actin cytoskeleton. In addition to uncovering a long sought biological function for Old Yellow Enzyme, these results establish that cellular sensitivity to oxidative stress can in part be directly attributed to a specific form (C285-C374 disulfide bond formation) of oxidative damage to actin.     

Functional nonequivalency of actin isovariants in Arabidopsis.

Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenic Arabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.     

Synthetic study on the structure-activity relationship of sperm activating peptides from the jelly coat of sea urchin eggs

Various analogue peptides with substitution and deletion of amino acid residues have been synthesized by liquid phase method for Sperm Activating Peptides from the jelly coat of sea urchin eggs. The deletion of C-terminal Gly reduced the activity to about 1/3000, while removal of N-terminal Gly reduced the activity to 1/10. The residues Ser5 and Asp3 were replaced by Lys without significant loss of activity. Substitution of Phe2 by Gly, Ala or Pro markedly reduced the activity by the factor of 10(4)-10(6), in contrast to Tyr-substitution retaining almost full activity, indicating the essential role of the aromatic residue in exerting the activity. Substitutions, Asp3 to Glu and Gly10 to Pro, increased the activity 5-fold and 500-fold, respectively.