细胞色素c突变体的共振拉曼光谱研究

采用共振拉曼光谱技术研究了细胞色素c一次突变体（WT）及其突变体Y67F和N52I在低频区的光谱特征。结果表明，以苯丙氨酸替代WT中酪氨酸残基Tyr67并没有明显影响血红素丙氨酸侧基周围多肽氨基酸残基的构象，而异亮氨酸对天冬酰胺残基Asn52的取代则较大程度地改变了蛋白质内部水分子与周围氨基酸残基间的氢键作用和多肽空腔的疏水性，进而使氨基酸残基和血素的构象相应发生调变。两种取代都导致形成血红素周围空腔的多肽氨基酸残基构象的变化。     

Ultraviolet resonance Raman evidence for utilization of the heme 6-propionate hydrogen-bond network in signal transmission from heme to protein in Ec DOS protein

The direct oxygen sensor protein from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of either O2 or CO molecule to a reduced heme enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report ultraviolet resonance Raman (UVRR) spectroscopic investigations of the reduced, O2- and CO-bound forms of heme-bound PAS domain of Ec DOS. The UVRR results show that heme discriminates different ligands, resulting in altered conformations in the protein moiety. Specifically, the environment around Trp53 that contacts the 2-vinyl group of heme, is changed to a more hydrophobic environment by O2 binding, whereas it is changed to a more hydrophilic environment by CO-binding. In addition, the PDE activity of the O2- and CO-bound forms for the Trp53Phe mutant is significantly decreased compared with that of the wild type (WT), demonstrating the importance of Trp53 for the catalytic reaction. On the other hand, the binding of O2 or CO to the heme produces drastic changes in the Tyr126 of Ibeta-strand at the surface of the sensor domain. Furthermore, we found that Asn84 forms a hydrogen bond with Tyr126 either in the O2- or CO-bound forms but not in the reduced form. Finally, the PDE activities of the ligand-bound forms for Asn84Val and Tyr126Phe mutants are significantly reduced as compared with that of WT, suggesting the importance of the hydrogen-bonding network from heme 6-propionate to Tyr126 through Asn84 in signal transmission.     

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.     

Effect of side chains on competing pathways for beta-scission reactions of peptide-backbone alkoxyl radicals

High-level quantum chemistry calculations have been carried out to investigate beta-scission reactions of alkoxyl radicals located at the alpha-carbon of a peptide backbone. This type of alkoxyl radical may undergo three possible beta-scission reactions, namely C-C beta-scission of the backbone, C-N beta-scission of the backbone, and C-R beta-scission of the side chain. We find that the rates for the C-C beta-scission reactions are all very fast, with rate constants of the order 10(12) s(-1) that are essentially independent of the side chain. The C-N beta-scission reactions are all slow, with rate constants that range from 10(-0.7) to 10(-4.5) s(-1). The rates of the C-R beta-scission reactions depend on the side chain and range from moderately fast (10(7) s(-1)) to very fast (10(12) s(-1)). The rates of the C-R beta-scission reactions correlate well with the relative stabilities of the resultant side-chain product radicals (*R), as reflected in calculated radical stabilization energies (RSEs). The order of stabilities for the side-chain fragment radicals for the natural amino acids is found to be Ala < Glu < Gln approximately Leu approximately Met approximately Lys approximately Arg < Asp approximately Ile approximately Asn approximately Val < Ser approximately Thr approximately Cys < Phe approximately Tyr approximately His approximately Trp. We predict that for side-chain C-R beta-scission reactions to effectively compete with the backbone C-C beta-scission reactions, the side-chain fragment radicals would generally need an RSE greater than approximately 30 kJ mol(-1). Thus, the residues that may lead to competitive side-chain beta-scission reactions are Ser, Thr, Cys, Phe, Tyr, His, and Trp.     

Calculation of relative binding affinities of fructose 1,6-bisphosphatase mutants with adenosine monophosphate using free energy perturbation method

The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between inhibitors and protein variants. In this article, the importance of hydrophobic and hydrophilic residues to the binding of adenosine monophosphate (AMP) to the fructose 1,6-bisphosphatase (FBPase), a target enzyme for type-II diabetes, was examined by FEP method. Five mutations were made to the FBPase enzyme with AMP inhibitor bound: 113Tyr --> 113Phe, 31Thr --> 31Ala, 31Thr --> 31Ser, 177Met --> 177Ala, and 30Leu --> 30Phe. These mutations test the strength of hydrogen bonds and van der Waals interactions between the ligand and enzyme. The calculated relative free energies indicated that: 113Tyr and 31Thr play an important role, each via two hydrogen bonds affecting the binding affinity of inhibitor AMP to FBPase, and any changes in these hydrogen bonds due to mutations on the protein will have significant effect on the binding affinity of AMP to FBPase, consistent to experimental results. Also, the free energy calculations clearly show that the hydrophilic interactions are more important than the hydrophobic interactions of the binding pocket of FBPase.     

PHEOPHORBIDE a-INDUCED PHOTO-OXIDATION OF CYTOCHROME c: IMPLICATION FOR PHOTODYNAMIC THERAPY

Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

Role of Met93 and Thr96 in the Lid Hinge Region of Rhizopus chinensis Lipase

We engineered Rhizopus chinensis lipase to study its critical amino acid role in catalytic properties. Based on the amino acid sequence and three-dimensional model of the lipase, residues located in its lid hinge region (Met93 and Thr96) were replaced with corresponding amino acid residues (Ile93 and Asn96) found in the lid hinge region of Rhizopus oryzae lipase. The substitutions in the lid hinge region affected not only substrate specificity but also the thermostability of the lipase. Both lipases preferred p-nitrophenyl laurate and glyceryl trilaurate (C12). However, the variant S4-3O showed a slight decline in activity toward long-chain fatty acid (C16–C18). When enzymes activities decreased by half, the temperature of the variant (45 °C) was 22 °C lower than the parent (67 °C), probably substantially destabilized the structure of the lid region. The interfacial kinetic analysis of S4-3O suggested that the lower catalytic efficiency was due to a higher K _m* value. According to the lipase structure investigated, Ile93Met played a role of narrowing the size of the hydrophobic patch, which affected the substrate binding affinity, and Asn96Thr destabilized the structure of the lipase by disrupting the H-bond interaction in the lid region.     

白色念珠菌N-肉豆蔻酰基转移酶中药物结合位点的MCSS分析

采用多拷贝同时搜寻方法(MCSS)分析得到了CaNMT活性位点的疏水区域、氢键结合位点和负电性区域. MCSS计算结果显示, CaNMT活性位点有两个疏水性比较强的区域: 一个由Tyr107, Tyr109, Val108, Phe117, Phe123, Ala127, Phe176和Leu337等残基组成; 另一个由Phe115, Phe240和Phe339组成. CaNMT活性位点发现有两个氢键作用区域, 其中Tyr119, His227, Asn392和Leu451是与已有抑制剂的氢键结合位点, Tyr107, Asn175, Thr211和Asp412是新发现的氢键结合位点, 而且在NMT家族中高度稳定, 它们对设计新结构类型的CaNMT抑制剂具有重要作用. Leu451是负电性兼氢键作用位点, 是抑制剂设计时所必需考虑的位点.     

Direct electrochemistry of cytochrome c on nanotextured diamond surface

Nanotextured diamond surfaces with geometrical properties close to protein dimensions were used for the realization of direct electron transfer of cytochrome c (cyt c) without any covalent bonding. The peroxidase activity of native and denatured cyt c was also investigated. Cyclic voltammograms of native cyt c show quasi-reversible electron transfer reactions, while no heme redox activity is detected for denatured cyt c. Unfolding (denaturation) of cyt c can be achieved in the presence of hydrogen peroxide. Partially or fully denatured cyt c showed higher peroxidase activity than native cyt c. This is because denatured cyt c loses its tertiary structure and hydrogen peroxide is easier to access the heme redox center. The apparent Michaelis–Menten constant K_m for native and denatured cyt c has been determined to be 0.23 mM and 0.08 mM.     

Comparative study of the inactivation of horseradish peroxidase under the effect of H2O2 and ionizing radiation

The inactivation of native and recombinant horseradish peroxidase in the presence of hydrogen peroxide and under ionizing radiation was studied. The types of peroxidase activity differ in sensitivity towards the inactivating effect of H₂O₂: the activity in relation to the iodide ion is more stable than the activity in relation to ammonium 2,2-azinobis(3ethylbenzothiazoline-6-sulfonate) (ABTS) ando-phenylenediamine. Similar inactivation was observed in the course of the radiolysis of peroxidase. It was assumed that the initial period of peroxidase inactivation in the presence of hydrogen peroxide has a radical nature and is related to the generation of Superoxide radicals, which modify the protein moiety, resulting in the destruction of heme. The R-670 compound was not formed under the conditions studied. However, the E EI transition occurred, depending on the radiation dose and the enzyme concentration.Translated fromIzyestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 176–179, January, 1995.     

A surfactant–heme–sulfonyl imidazole system as a nano-artificial enzyme

The heme–imidazole–sodium dodecyl sulfate (SDS) ternary complex has been designed as a peroxidase-like nano-artificial enzyme, in which the imidazole moiety functions like the histidine ligand in the native horseradish peroxidase (HRP) and increases the reactivity and catalytic efficiency of the designed artificial enzyme by promoting the heterolytic cleavage of hydrogen peroxide. In the present study, three different ligands were used as the imidazole-based ligands in the heme–ligand–SDS ternary system: (1) 1-methylsulfonyl-1H-imidazole, (2) 1-(benzensulfonyl)-1H-imidazole, and (3) 1-tosyl-1H-imidazole (TsIm). The three different ligands gave variable reactivity in the system studied, and the enzymatic activation parameters, using spectrophotometric measurements, showed that the TsIm ligand had a higher catalytic efficiency at 26.38 % of the native HRP efficiency. To investigate the increase in catalytic activity, its mechanism was explored based on the original mechanism of HRP and the structure of its first catalytic intermediate (compound I). Based on the mechanism of HRP and the structure of compound I, a suggested mechanism for Tslm is as follows: the TsIm cation radical makes up part of the compound I structure, which is stabilized in the enzymatic process by charge distribution that is induced via phenyl and methyl groups. Suicide inactivation of heme–TsIm–SDS and heme–imidazole–SDS models was also compared to each other. Suicide inactivation was less exhibited in the presence of TsIm than imidazole in this system unless high concentrations of hydrogen peroxide were used.     

Mechanistic insights into mode of action of rice allene oxide synthase on hydroxyperoxides: An intermediate step in herbivory-induced jasmonate pathway

Various types of oxygenated fatty acids termed ‘oxylipins’ are involved in plant response to herbivory. Oxylipins like jasmonic acid (JA) and green leafy volatiles (GLVs) are formed by the action of enzymes like allene oxide synthase (AOS) and hydroxyperoxide lyase (HPL) respectively. In this study, we focus on AOS of Oryza sativa sb. Japonica, that interact with 9- and 13- hydroxyperoxides to produce intermediates of jasmonate pathway and compare it with rice HPL that yields GLVs. We attempt to elucidate the interaction pattern by computational docking protocols keeping the Arabidopsis AOS system as the reference model system. Both 9-hydroxyperoxide and 13-hydroxyperoxide fit into the active site of AOS completely with Phe347, Phe92, Ile463, Val345, and Asn278 being the common interacting residues. Phe347 and Phe92 were mutated with Leucine and docked again with the hydroxyperoxides. The Phe347 → Leu347 mutant showed a different mode of action than AOS-hydroxyperoxide complex with Trp413 in direct bonding with the OOH group of 9-hydroxyperoxide. The loss of Lys88-OOH interaction in 13-hydroxyperoxide and loss-of-interaction of Leu347 indicated the importance of Phe347 residue in hydroxyperoxide catalysis. The second mutant Phe92 → Leu92 also shows a very different interaction pattern with 13-hydroxyperoxide but not with 9-hydroxyperoxide.Therefore, it can be concluded that Phe347 is more crucial for AOS functionality than Phe92. The aromatic ring of a Phenylalanine residue is important for catalysis and its mutation affects the binding of the two ligands. Another important residue is Asn278 which is an important part of the AOS catalytic site for maintaining stability and can be compared with the Arabidopsis AOS residue Asn321. Lastly, the interaction of HPL with these two derivatives involves Leu363 residue instead of Phe347 and thus, validating the importance of Phe → Leu substitution to be the reason of different modes of action that result in completely different products from same substrates.     

Effective synthesis of cyclic peptide yunnanin C and analogues via Ser/Thr ligation (STL)-mediated peptide cyclization

Cyclic peptide yunnanin C isolated from the root of Stellaria yunnanensis was efficiently synthesized in which the linear peptide was prepared by Boc-SPPS and the cyclization was realized by serine/threonine ligation (STL)-mediated cyclization. In addition, nine yunnanin C analogues, including mutations of Tyr7Gly, Tyr7Val, Tyr7Pro, Tyr7Phe, Ser1Thr, Pro2Val, Gly5Pro, Phe6Ala and Ile4Ala, were prepared in the same fashion. Here, we demonstrated that STL-mediated peptide cyclization could be an effective approach to construct cyclic peptides. Except that proline at the C-terminus could retard the cyclization process, cyclization of yunnanin C analogues with various C-terminal amino acids proceeded with fast cyclization rate (<4 h) and only trace amount of dimers (<5%) at a working concentration of 5 mM.     

游离芳香氨基酸和半胱氨酸对光诱导高铁肌红蛋白还原的影响

肌红蛋白(Myoglobin,Mb)中血红素辅基不仅具有储氧功能,也能吸收特定波长的光而影响蛋白功能表达。实验发现,部分游离的氨基酸对光诱导高铁肌红蛋白(metM b,Fe(III)-Mb)的还原过程及还原程度都有重要作用,因此,本文采用紫外-可见吸收光谱、圆二色谱、三维荧光光谱法,在光照体系中加入拥挤试剂来模拟细胞内拥挤环境,研究芳香氨基酸[色氨酸(Trp)、苯丙氨酸(Phe)、酪氨酸(Tyr)、半胱氨酸(Cys)]对metM b还原的影响。结果表明,含-OH或-SH的氨基酸(Tyr、Cys)能使metM b发生较好的光还原,无-OH或-SH基团的氨基酸(Trp、Phe)对metM b的光还原作用较弱,氨基酸促进metM b光还原的整个过程可能是分子间电子转移的过程。metM b在拥挤环境聚蔗糖70(Ficoll 70)中的光诱导还原程度比在稀溶液中高,拥挤试剂Ficoll70对蛋白的二级结构起保护作用,能够稳定血红素微环境。     

THE DISTRIBUTION FUNCTION OF RESIDUE-RESIDUE CONTACTS IN PROTEIN MOLECULES

In protein molecules each residue has a different ability to form contacts.In this paper,we calculated the number of contacts per residue and investigated the distribution of residue-residue contacts from 495 globular protein molecules using Contacts of Structural Units(CSU)software.It was found that the probability P(n)of amino acid residues having n pairs of contacts in all contacts fits Gaussian distribution very well.The distribution function of residue-residue contacts can be expressed as:P(n)=P_0+aexp[-b(n-n_c)~2].In our calculation,P_0=-0.06,α=11.4,b=-0.04 and n_c=9.0.According to distribution function,we found that those hydrophobic(H)residues including Leu,Val,Ile,Met,Phe,Tyr,Cys,and Trp residues have large values of the most probable number of contact n_c,and hydrophilic(P)residues including Ala,Gly,Thr, His,Glu,Gln,Asp,Asn,Lys,Ser,Arg,and Pro residues have the small ones.We also compare with Fauchere-Pliska hydrophobicity scale(FPH)and the most probable number of contact n_c for 20 amino acid residues,and find that there exists a linear relationship between Fauchere-Pliska hydrophobicity scale(FPH)and the most probable number of contact n_c, and it is expressed as:n_c=a+b×FPH,here α=8.87,and b=1.15.It is important to further explain protein folding and its stability from residue-residue contacts.     

Trypsin inhibiting material from Ascaris lumbricoides var. suum. I. Preparation of pure trypsin inhibitors

The purification of a trypsin inhibitor from Ascaris lumbricoides var. suum is described. The electrophoretically pure preparation which inhibits trypsin in a specific manner is a relatively small peptide containing 5 Asp, 4 Thr, 1 Ser, 11 Glu, 6 Pro, 6 Gly, 5 Ala, 2 Val, 10 (Cys)_1/2, 3 Ile, 2 Phe, 7 Lys, 3 Arg and 1 Try.     

Hydroperoxynaphthalimide Derivative-mediated Oxidation of Heme Proteins on Photoirradiation: Horseradish Peroxidase

Abstract— Horseradish peroxidase (HRP) was photoirradiated in the presence of organic peroxide (1, hydroperoxynaphthalimide derivative) at around 353 nm and 0°C. This compound bound to a heme pocket of HRP as shown by its inhibitory effect on catalysis by HRP ( K _i= 5.5 times 10⁻⁵ M) and subsequently it formed an intermediate in the same way as H202. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggested cleavage of the peptide chain of HRP on photoirradiation with 1. From CD spectra and SDS-PAGE, it was presumed that the destruction of both secondary structure and heme of the enzyme occurred to some extent upon photoirradiation, which resulted in a decrease in the catalytic activity. The absorption spectra also suggested that the heme group of the enzyme was destroyed, and the fluorescence spectra showed that the Trp residue in the photoirradiated HRP was oxidized to N -formylkynurenine by a hydroxyl radical generated from 1. Energy transfer from the excited naphthalimide moiety or hydrogen abstraction also seemed to make some contribution to the alteration of the heme group.     

Oxygen activation and electron transfer in flavocytochrome P450 BM3

In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.     

嗜热蛋白酶PH1704别构中心量子化学计算与突变体动力学简

通过量子化学计算,确定嗜热菌Pyrococcus horikoshii OT3的PH1704蛋白酶别构位点的关键残基为Arg113,Tyr120和Asn129. 其中,Arg113及Asn129与别构抑制剂结合,参与别构调控. Tyr120残基位于亚基交界面附近,并与亲核残基Cys100之间以氢键相连,可通过影响亚基聚合来影响酶的亲核催化. DJ-1超家族的4种构建蛋白的结构显示,120位点位于亚基交界面处,影响亚基的聚合,进而影响蛋白酶的活力,并间接参与别构调控. 分子生物学实验显示,突变体R113T/Y120P/N129D的kcat/km(L·μmol-1·min-1)值是野生型kcat/km值的6倍,h系数由野生型的0.86转变为1.3,负协同效应消失. 113和129位点处阴离子别构剂脱离,从而破坏113,120和129位点间的封闭环结构,使AC交界面α7螺旋(124~129,524~529)间聚合度增强;120位点残基由Tyr转变为Pro,与Cys100间氢键断裂,亲核进攻的阻力减小,从而使酶活力提高,别构负调控消失.