Mass spectrometry of hydrogen/deuterium exchange of Escherichia coli dihydrofolate reductase: effects of loop mutations

To address the effects of single amino acid substitutions on the structural fluctuation of Escherichia coli dihydrofolate reductase (DHFR), hydrogen/deuterium exchange kinetics were investigated at 15 degrees C with wild-type and mutant DHFRs at Gly67 (six mutants) and Gly121 (eight mutants) located in two flexible loops, by means of electrospray ionization mass spectrometry. These mutations induced significant changes in the first-order rate constant of proton exchange, k(ex) (0.10-0.27 min(-1)), the number of fast-exchangeable protons, Delta M(o) (164-222 Da), and the number of protons protected from exchange, Delta M(infinity) (15-56 Da), relative to the corresponding values for the wild-type enzyme (k(ex) = 0.18 min(-1), Delta M(o) = 164 Da, and Delta M(infinity) = 50.5 Da). These kinetic parameters were strongly correlated with the volume of introduced amino acids, but partly correlated with adiabatic compressibility (volume fluctuation), stability, and enzymatic activity. These results indicate that the local structure change due to a single amino acid substitution in loop regions is dramatically magnified to affect the structural fluctuation of the whole DHFR molecule, resulting in complicated changes in its stability and function.     

A linear correlation between the energetics of allosteric communication and protein flexibility in the Escherichia coli cyclic AMP receptor protein revealed by mutation-induced changes in compressibility and amide hydrogen-deuterium exchange

Amino acid substitutions at distant sites in the Escherichia coli cyclic AMP receptor protein (CRP) have been shown to affect both the nature and magnitude of the energetics of cooperativity of cAMP binding, ranging from negative to positive. In addition, the binding to DNA is concomitantly affected. To correlate the effects of amino acid substitutions on the functional energetics and global structural properties in CRP, the partial specific volume (v(o)), the coefficient of adiabatic compressibility (beta(s)(o)), and the rate of amide proton exchange were determined for the wild-type and eight mutant CRPs (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) by using sound velocity, density measurements, and hydrogen-deuterium exchange as monitored by Fourier transform infrared spectroscopy at 25 degrees C. These mutations induced large changes in v(o) (0.747-0.756 mL/g) and beta(s)(o) (6.89-9.68 Mbar(-1)) compared to the corresponding values for wild-type CRP (v(o)= 0.750 mL/g and beta(s)(o)= 7.98 Mbar(-1)). These changes in global structural properties correlated with the rate of amide proton exchange. A linear correlation was established between beta(s)(o) and the energetics of cooperativity of binding of cAMP to the high-affinity sites, regardless of the nature of cooperativity, be it negative or positive. This linear correlation indicates that the nature and magnitude of cooperativity are a continuum. A similar linear correlation was established between compressibility and DNA binding affinity. In addition, linear correlations were also found among the dynamics of CRP and functional energetics. Double mutation (K52N/H159L) at positions 52 and 159, whose alpha-carbons are separated by 34.6 A, showed nonadditive effects on v(o) and beta(s)(o). These results demonstrate that a small alteration in the local structure due to amino acid substitution is dramatically magnified in the overall protein dynamics which plays an important role in modulating the allosteric behavior of CRP.     

Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility

The partial specific volume, v, and adiabatic compressibility, beta(s), of Escherichia coli dihydrofolate reductase were measured at 30 degrees C in the presence of various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP, methotrexate, and KCl). Binding of these ligands (binary and ternary complexes) brought about large changes of v (0.734-0.754 cm(3) g(-1)) and beta(s) (6. 6x10(-6)-9.8x10(-6) bar(-1)), keeping a linear relationship between the two parameters. The values of v and beta(s) increased with an increase in internal cavity, V(cav), and a decrease in accessible surface area, ASA, which were calculated from the X-ray crystal structures of the complexes. A large variation of V(cav) relative to ASA by ligand binding suggested that the cavity is a dominant factor and the effect of hydration might be small for the ligand-induced changes of v and beta(s). The beta(s) values of the binary and ternary complexes suggested a characteristic conformational flexibility of the kinetic intermediates in the enzyme reaction coordinate. Comparison of beta(s) with the cavity distribution in the crystal structures revealed that the flexibility of the intermediates was mainly determined by the total cavity volume with minor contributions of the number, position, and size of cavities. These results demonstrate that the compressibility is a useful measure of the conformational flexibility of the intermediates in the enzyme reaction and that the combined study of compressibility and X-ray crystallography gives new insight into the protein dynamics through the behavior of the cavities.     

Volumetric and spectroscopic characterizations of the native and acid-induced denatured states of staphylococcal nuclease

We have characterized the acid-induced denaturation of staphylococcal nuclease (SNase) at different urea concentrations by a combination of ultrasonic velocimetry, high precision densimetry, and CD spectroscopy. Our CD spectroscopic results suggest that, at low salt and acidic pH, the protein is unfolded with disrupted secondary and tertiary structures. Furthermore, as judged by far UV CD spectra, the protein is further unfolded at acidic pH upon the addition of urea up to the concentration of 1.5 M. The midpoint of the transition shifts to more neutral pH values and the cooperativity of the transition decreases as the acid-induced denaturation of SNase occurs at higher urea concentrations. We find that the change in volume, Deltav, accompanying the acid-induced denaturation of SNase increases from -0.013 cm(3) g(-1) (-218 cm(3) mol(-1)) in the absence of urea to 0.011 cm(3) g(-1) (185 cm(3) mol(-1)) at 1.5 M urea. At all urea concentrations, the partial specific adiabatic compressibility, k(o)(s), of the protein decreases upon its unfolding with the values of Deltak(o)(s) equal to -6.3x10(-6) (-0.106 cm(3) mol(-1) bar(-1)), -4.5x10(-6) (-0.076 cm(3) mol(-1) bar(-1)), -4.6x10(-6) (-0.077 cm(3) mol(-1) bar(-1)), and -3.8x10(-6) (-0.064 cm(3) mol(-1) bar(-1)) cm(3) g(-1) bar(-1) at urea concentrations of 0, 0.5, 1.0, and 1.5 M, respectively. In general, our volumetric results suggest that the acid-induced denatured state of SNase is only partially unfolded with the solvent-exposed surface area equal to 70-80 % of that expected for the fully extended conformation.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

Role of the covalent glutamic acid 242-heme linkage in the formation and reactivity of redox intermediates of human myeloperoxidase

In human myeloperoxidase the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl group on pyrrole ring A. In the present study, wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln were produced in Chinese hamster ovary cells and investigated in a comparative sequential-mixing stopped-flow study in order to elucidate the role of the Glu242-heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the ester bond increased heme flexibility, blue shifted the UV-vis spectrum, and, compared with recMPO, decelerated cyanide binding (1.25 x 10(4) versus 1.6 x 10(6) M(-)(1) s(-)(1) at pH 7 and 25 degrees C) as well as compound I formation mediated by either hydrogen peroxide (7.8 x 10(5) versus 1.9 x 10(7) M(-)(1) s(-)(1)) or hypochlorous acid (7.5 x 10(5) versus 2.3 x 10(7) M(-)(1) s(-)(1)). The overall chlorination and bromination activity of Glu242Gln was 2.0% and 24% of recMPO. The apparent bimolecular rate constants of compound I reduction by chloride (65 M(-)(1) s(-)(1)), bromide (5.4 x 10(4) M(-)(1) s(-)(1)), iodide (6.4 x 10(5) M(-)(1) s(-)(1)), and thiocyanate (2.2 x10(5) M(-)(1) s(-)(1)) were 500, 25, 21, and 63 times decreased compared with recMPO. By contrast, Glu242Gln compound I reduction by tyrosine was only 5.4 times decreased, whereas tyrosine-mediated compound II reduction was 60 times slower compared with recMPO. The effects of exchange of Glu242 on electron transfer reactions are discussed.     

脱落酸对低温下雷公藤幼苗光合作用及叶绿素荧光的影响

以1年生雷公藤扦插苗为试材,研究低温胁迫下不同浓度外源脱落酸（ABA,0、5、10、15、20、25 mg·L^-1)叶面喷施处理对雷公藤叶片光合作用及叶绿素荧光参数的影响.结果表明：喷施20 mg·L^-1的ABA能显著提高雷公藤幼苗的抗冷性,减缓低温下雷公藤叶片净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(g_s)、胞间CO₂浓度(C_i)的下降幅度,提高幼苗叶片的光合能力.低温处理6 d后,随着ABA浓度上升,雷公藤叶片的初始荧光(F_o)下降,最大荧光(F_m)和PSII最大光化学效率(F_v/F_m)上升,PSII实际光化学量子产量(Φ_PSⅡ)、光化学猝灭系数(q_P)先下降后上升,而非光化学猝灭系数(q_N)呈下降－上升－下降趋势.P_n、g_s、q_P、F_m和F_v/F_m均在20 mg·L^-1ABA处理时达到峰值.不同浓度ABA的相对电子传递速率(rETR)随着光化光强度增加呈先上升后下降的趋势,当光化光强度(PAR)达到395 μmol·m^-2s^-1时,各处理的rETR达到最高值,其中25 mg·L^-1和20 mg·L^-1ABA处理分别比对照高17.1%和5.2%.雷公藤叶片Φ_PSⅡ的光响应曲线均随光化光强度升高而下降,q_N的光响应曲线则呈相反趋势.     

Synthetic, biologically active amphiphilic peptides

Amphiphilic peptides typically consist of a peptide portion that may be 5-25 (or more) amino acids in length. The hydrophobic portion may be a single fatty acid residue, but can also be more elaborate. The main focus of this article lies on the family of synthetic anion binders (SATs) of the general structure (R(1))(2)N-COCH(2)OCH(2)CO-(Aaa)(n)-OR(3). The most-common R(1) group is the octadecyl (C(18)H(37)) group. The most studied peptide sequence in this family is (Gly)(3)-Pro-(Gly)(3), although different sequences (and longer and shorter peptides) have been prepared as well. The C-terminal ester residue providing the most effective anion release from liposomes is heptyl (C(7)H(15)), although many others have been examined. The compound (C(18)H(37))(2)N-COCH(2)OCH(2)CO-(Gly)(3)-Pro-(Gly)(3)-OBn (Bn=benzyl) was found to mediate Cl(-) transport in mouse epithelial cells.     

Partial identification of carbohydrate-binding sites of a Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three identical subdomains (alpha, beta, and gamma) and a C-terminal domain of unknown function. Here, we investigate the structure-function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln(46) in the alpha subdomain and Trp(138) in the gamma subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOADeltaC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A) was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of the wild-type MOA. On the basis of this observation, we conclude that both alpha and gamma subdomains are most probably involved in carbohydrate binding, but the beta subdomain appears to be inactive.     

Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter

The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG). In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B(1), A(2), and B(2) motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V(max) but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity. Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled. Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.     

Site-directed deletion mutants of a carboxyl-terminal region of human dihydrofolate reductase.

Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35% decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33% that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38% wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild-type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis.     

Interconversion of Cu(I) and Cu(II) forms of galactose oxidase: comparison of reduction potentials

A procedure for the preparation of the fully reduced Cu(I) form of galactose oxidase, GOase(red), involving reduction of GOase(semi) (or GOase(ox)) with non-coordinating [Ru(NH(3))(6)](2+) (51 mV vs. nhe) is described. Air-free conditions and a two-fold excess of [Ru(NH(3))(6)](2+) give a stable product with no further UV-Vis changes over >1.5 h. Rate constants for the reduction of GOase(semi) (k(f)=860 M(-1) s(-1)) give a first-order [H(+)]-dependence (pK(1a)=7.9), but the reverse process involving [Ru(NH(3))(6)](3+) oxidation of GOase(red) (k(b)=18.6 M(-1) s(-1)) is independent of pH (5.5 to 9.5). The reduction potential E(2)(o)' (vs. nhe) for the GOase(semi)/GOase(red) (i.e. Cu(II)/Cu(I)) couple is 149 mV at pH 7.5, which varies from 160 mV (pH 5.5) to 120 mV (pH 10.5), suggesting pK(1a) (GOase(semi)) and pK(2a) (GOase(red)) acid dissociation constants both involving Tyr-495. It is concluded that pK(2a) is for acid dissociation of uncoordinated H(+)Tyr-495. Consistent with this interpretation rate constants/M(-1) s(-1) for the GOase(semi) Tyr495 Phe variant, k(f)=1.59x10(3) and k(b)=16.1, respectively, are independent of pH and give a reduction potential of 169 mV. Comparisons are made of reduction potentials (E(1)(o)'/mV pH 7.5) for the GOase(ox)/GOase(semi) (i.e. Tyr(.)/Tyr) couple, and are for the Cys228Gly variant (630), for enzyme with N(3)(-) for H(2)O at the substrate binding exogenous site (393), and for apo-protein (570). These compare with previously reported values for the variants Trp290His (730) and Tyr495Phe (450), and together serve to quantify different contributions to the unusually small E(1)(o)' of 400 mV for the Tyr(.)/Tyr couple. At pH 7.5 the reduction potential for the two-equivalent GOase(ox)/GOase(red) couple is calculated to be 275 mV. The rate constant for the reaction of GOase(red) with GOase(ox) is 4.4x10(3) M(-1) s(-1) at pH 7.5.     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Energetics of kayaking at submaximal and maximal speeds

The energy cost of kayaking per unit distance (C(k), kJ x m(-1)) was assessed in eight middle- to high-class athletes (three males and five females; 45-76 kg body mass; 1.50-1.88 m height; 15-32 years of age) at submaximal and maximal speeds. At submaximal speeds, C(k) was measured by dividing the steady-state oxygen consumption (VO(2), l x s(-1)) by the speed (v, m x s(-1)), assuming an energy equivalent of 20.9 kJ x l O(-1)(2). At maximal speeds, C(k) was calculated from the ratio of the total metabolic energy expenditure (E, kJ) to the distance (d, m). E was assumed to be the sum of three terms, as originally proposed by Wilkie (1980): E = AnS + alphaVO(2max) x t-alphaVO(2max) x tau(1-e(-t x tau(-1))), were alpha is the energy equivalent of O(2) (20.9 kJ x l O(2)(-1)), tau is the time constant with which VO(2max) is attained at the onset of exercise at the muscular level, AnS is the amount of energy derived from anaerobic energy utilization, t is the performance time, and VO(2max) is the net maximal VO(2). Individual VO(2max) was obtained from the VO(2) measured during the last minute of the 1000-m or 2000-m maximal run. The average metabolic power output (E, kW) amounted to 141% and 102% of the individual maximal aerobic power (VO(2max)) from the shortest (250 m) to the longest (2000 m) distance, respectively. The average (SD) power provided by oxidative processes increased with the distance covered [from 0.64 (0.14) kW at 250 m to 1.02 (0.31) kW at 2000 m], whereas that provided by anaerobic sources showed the opposite trend. The net C(k) was a continuous power function of the speed over the entire range of velocities from 2.88 to 4.45 m x s(-1): C(k) = 0.02 x v(2.26) (r = 0.937, n = 32).     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus

The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a beta-barrel structure formed from seven anti-parallel beta-strands in two beta-sheets. One anti-parallel beta-sheet consists of beta-strands beta1 (Phe(299)-Asp(307)), beta2 (Val(313)-Tyr(319)), beta4 (Arg(354)-Leu(355)), and beta5 (Lys(370)-Glu(376)) arranged so that beta2 is flanked on either side by beta1 and beta5. The short beta4 flanks the end of the remaining side of beta5. The remaining anti-parallel beta-sheet is formed from strands beta3 (Ile(340)-Val(343)), beta6 (Gly(380)-Arg(388)), and beta7 (Gln(391)-Lys(399)) arranged with beta6 at the center. Residues implicated in antigenic differences between different West Nile virus strains (and other flaviviruses) and neutralization are located on the outer surface of the protein. Characterization of the binding of monoclonal antibodies to WN-rED3 mutants, which were identified through neutralization escape experiments, indicate that antibody neutralization directly correlates with binding affinities. These studies provide an insight into theoretical virus-receptor interaction points, structure of immunogenic determinants, and potential targets for antiviral agents against West Nile virus and highlight differences between West Nile virus and other flavivirus structures that may represent critical determinants of virulence.     

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.     

Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.