The Experts below are selected from a list of 2043 Experts worldwide ranked by ideXlab platform
Ulrich Hahn - One of the best experts on this subject based on the ideXlab platform.
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Impact of four 13C-proline isotope labels on the infrared spectra of Ribonuclease T1
Journal of the American Chemical Society, 2002Co-Authors: Ralf Moritz, Ulrich Hahn, Heinz Fabian, Max Diem, Dieter NaumannAbstract:Ribonuclease T1 was biosynthesized, with all four prolines 13C-labeled in the peptide CO bond, using a proline auxotrophic yeast strain of Saccharomyces cerevisiae. The 13C- and 12C-proline isotopomers of Ribonuclease T1 were investigated by infrared spectroscopy in the thermally unfolded and natively folded state at 80 and 20 °C, respectively. In the thermally unfolded state, both proteins established almost indistinguishable spectral features in the secondary structure sensitive amide I region. In contrast, the spectra measured at 20 °C revealed substantial qualitative and quantitative differences, though parallel analysis by circular dichroism suggested identical native folds for both isotopomers. Major spectral differences in the infrared spectra were detected at 1626 and 1679 cm-1, which are diagnostic marker bands for antiparallel β-sheets in Ribonuclease T1 and at 1645 cm-1, a region that is characteristic for the infrared absorption of irregular structures. Starting with the known three-dimensiona...
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Ribonuclease T1 cleaves RNA after guanosines within single-stranded gaps of any length.
Nucleosides Nucleotides and Nucleic Acids, 2000Co-Authors: Thomas Greiner-stöffele, Hans-heinrich Foerster, Ulrich HahnAbstract:RNA-oligonucleotides with defined single-stranded stretches were designed to investigate the minimal requirements of a Ribonuclease T1 substrate. It could be shown, that RNase T1 cleaves single-stranded RNA after a unique guanosine flanked by two double-stranded areas. However, the turnover of such a G-gap is significantly lower than that of a gap of two, three or four nucleotides.
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Ribonuclease T1 has different dimensions in the thermally and chemically denatured states: a dynamic light scattering study
FEBS Letters, 1997Co-Authors: Klaus Gast, Ulrich Hahn, Matthias Wirth, Dietrich Zirwer, Hilde Damaschun, Marlies Müller-frohne, Gregor DamaschunAbstract:Abstract Ribonuclease T1 can be unfolded and refolded without forming noticeable amounts of aggregates allowing to characterise the dimensions of a protein in different denatured states in terms of the Stokes radius R S . Upon thermal unfolding R S increases from 1.74 nm at 20°C to 2.14 nm at 60°C. By contrast, R S =2.40 nm was obtained at 5.3 M guanidinium chloride (GuHCl) and 20°C. Heating from 20°C to 70°C in the presence of 5.3 M GuHCl led to a 5% decrease in R S . © 1997 Federation of European Biochemical Societies.
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Display Of Ribonuclease T1 On The Surface Of Bacteriophage M13
Nucleosides and Nucleotides, 1997Co-Authors: Bernd Hubner, Kerstin Korn, Hans-heinrich Förster, Ulrich HahnAbstract:Abstract In this work the expression of functional Ribonuclease T1 on the surface of the filamentous Escherichia coli phage M13 is described. Ribonuclease T1 was fused to the phage coat protein pIII and functionally displayed on the phage surface as shown by a negative staining zymogram and RNase indicator plates.
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Reverse action of Ribonuclease T1 in frozen aqueous systems.
Biological Chemistry, 1997Co-Authors: Marion Haensler, Ulrich Hahn, Hans-dieter JakubkeAbstract:We have studied Ribonuclease T1 (EC 3.1-27.3)-catalysed synthesis of guanylyl-(3'-->5')cytidine in frozen aqueous reaction mixtures at -10 degrees C and in solution at 0 degree C in order to investigate whether Ribonuclease-catalysed synthetic reactions can take advantage of the yield-increasing effect of freezing as was reported for protease-catalysed peptide synthesis. Under frozen state conditions, substantially increased yields of GpC were obtained compared to the reactions in solution. From the fact that no irreversible hydrolysis of the 2'3'-cyclic donor was observed it can be concluded that transesterification of the newly formed phosphodiester bond is the most important yield-limiting factor in Ribonuclease T1-catalysed dinucleoside phosphate synthesis.
Wolfram Saenger - One of the best experts on this subject based on the ideXlab platform.
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X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in Ribonuclease T1.
European Journal of Biochemistry, 1994Co-Authors: Wolf-dieter Schubert, Ulrich Hahn, Gerd Schluckebier, Jan Backmann, Joachim Granzin, Caroline Kisker, Hui-woog Choe, Wolfgang Pfeil, Wolfram SaengerAbstract:Two mutants of Ribonuclease T1 (RNaseT1), [59-tyrosine]Ribonuclease T1 (W59Y) and [45-tryptophan, 59-tyrosine]Ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the inhibitor 2′-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P21, with two molecules/asymmetric unit, W59Y: a= 4.934 nm, b= 4.820 nm, c= 4.025 nm, β= 90.29°. Y45W/W59Y: a= 4.915 nm, b= 4.815 nm, c= 4.015 nm, β= 90.35°. Compared to wild-type RNaseT1 in complex with 2′-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single α-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]Ribonuclease T1 (Y24W), [42-tryptophan]Ribonuclease T1 (Y42W), [45-tryptophan]Ribonuclease T1 (Y45W), [92-alanine]Ribonuclease T1 (H92A) and [92-threonine]Ribonuclease T1 (H92T) with and without the further mutation Trp59Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.
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The complex between Ribonuclease T1 and 3'GMP suggests geometry of enzymic reaction path. An X-ray study.
European Journal of Biochemistry, 1993Co-Authors: Andrea Heydenreich, Ulrich Hahn, Caroline Kisker, Hui-woog Choe, Gertraud Koellner, Frank Cordes, Hermann Schindelin, Ryszard W. Adamiak, Wolfram SaengerAbstract:The crystal structure of the complex between Ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of Ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of Ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.
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Modes of mononucleotide binding to Ribonuclease T1.
The Journal of biological chemistry, 1992Co-Authors: Yannis Georgalis, Ulrich Hahn, Athina Zouni, Piotr Zielenkiewicz, J F Holzwarth, Ronald J. Clarke, Wolfram SaengerAbstract:Abstract The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered Ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of Ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.
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Evidence for a substrate-binding subsite in Ribonuclease T1. Crystal structure of the complex with two guanosines, and model building of the complex with the substrate guanylyl-3',5'-guanosine.
Journal of Biological Chemistry, 1991Co-Authors: Andrea Lenz, Udo Heinemann, F Cordes, Wolfram SaengerAbstract:Abstract The enzyme Ribonuclease T1 cleaves single-stranded RNA at the 3'-side of guanosine. The structure of the complex with two guanosines has been analyzed at 1.8-A resolution and refined to a crystallographic R value of 14.0%. One guanosine occupies the guanosine recognition site as observed in previously analyzed complexes of Ribonuclease T1 with guanosine phosphates. The other is bound to a base-unspecific subsite marking the binding locus of the nucleoside 3'-proximal to guanosine in a cleavable RNA chain. The positions of the guanosine bound to the recognition site and of the guanine base at the subsite were used to guide model building of the substrate guanylyl-3',5'-guanosine bound to the active site of Ribonuclease T1. After energy minimization and a 7-ps stochastic dynamics simulation, a plausible model of the enzyme-substrate complex was obtained which may serve as a reference point in consideration of the mechanisms of RNA hydrolysis by Ribonuclease T1.
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Ribonuclease T1 struktur funktion und stabilitat
Angewandte Chemie, 1991Co-Authors: Nick C Pace, Ulrich Hahn, Udo Heinemann, Wolfram SaengerAbstract:In lebenden Organismen erfullen Proteine die wichtigsten und schwierigsten Aufgaben. Hierfur mussen sie oft spezifisch mit weiteren kleinen oder grosen Molekulen zusammenwirken. Dies erfordert, das sie sich in eine globulare Struktur falten, in der sie ihre jeweilige Funktion erfullen konnen; bei Enzymen z.B. wird durch die Faltung das aktive Zentrum konstituiert. Dieser enge Zusammenhang zwischen Struktur und Funktion fuhrte dazu, das Faltungsweise und Faltungsstruktur der Proteine als das „Geheimnis des Lebens” angesehen werden. Biochemiker und Chemiker haben ein groses Interesse an der Aufklarung des Mechanismus der Proteinfaltung. Ware das „Proteinfaltungsproblem” gelost, konnte man aus der Aminosauresequenz eines Proteins die bei der Faltung entstehende Struktur und ihre Stabilitat voraussagen. Es ist heute moglich, Proteine herzustellen, die sich in einer oder mehreren Aminosauren unterscheiden. Die Aufklarung ihrer dreidimensionalen Struktur durch Rontgenkristallographie und NMR-Spektroskopie tragt wesentlich zum Verstandnis der Faltung und der Stabilitat der Struktur von Proteinen bei. Aus diesem Grund werden gegenwartig mehrere Proteine mit dieser Methodik eingehend untersucht. Eines davon ist das Enzym Ribonuclease T1.
Franz X. Schmid - One of the best experts on this subject based on the ideXlab platform.
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a protein folding intermediate of Ribonuclease T1 characterized at high resolution by 1d and 2d real time nmr spectroscopy
Journal of Molecular Biology, 1999Co-Authors: Jochen Balbach, Clemens Steegborn, Thomas Schindler, Franz X. SchmidAbstract:Abstract The rate-limiting step during the refolding of S54G/P55N Ribonuclease T1 is determined by the slow trans→cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10°C. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the α-helix, β-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal β-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the α-helix, and the loops between β-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N Ribonuclease T1 and for cis/trans isomerizations are discussed.
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Catalyzed and assisted protein folding of Ribonuclease T1.
Biological chemistry, 1996Co-Authors: Franz X. Schmid, Christian Frech, Christian Scholz, Stefan WalterAbstract:The small single-domain protein Ribonuclease T1 (RNase T1) and variants thereof are good substrates for investigating the mechanisms of catalyzed and assisted protein folding. RNase T1 contains two cis prolines and two disulfide bonds, and the kinetic mechanism of its folding is well known. The wild-type form and designed variants that differ in the number prolines and of disulfide bonds were used as substrates to study the catalysis of folding by prolyl isomerases and protein disulfide isomerases. In its unfolded form, a marginally stable variant of RNase T1 binds to the chaperone GroEL and could thus be used to elucidate the kinetic mechanism of GroEL-mediated protein unfolding.
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Kinetic analysis of the unfolding and refolding of Ribonuclease T1 by a stopped-flow double-mixing technique.
Biochemistry, 1996Co-Authors: Lorenz M. Mayr, Christian Odefey, Mike Schutkowski, Franz X. SchmidAbstract:Often protein folding reactions show complex kinetics, because multiple unfolded species are present, which refold simultaneously. After conformational unfolding, these species are formed by the slow cis/trans equilibrations at Xaa-Pro peptide bonds. To dissect the roles of individual prolines for unfolding and refolding, we used Ribonuclease T1, a protein with two cis prolyl peptide bonds, preceding Pro39 and Pro55, and two variants with substitutions at these positions. A stopped-flow double-mixing technique was employed (i) to measure the rates of the individual prolyl isomerizations in the unfolded proteins and (ii) to study the refolding of transient species that are not well populated at equilibrium. In particular, the elusive species with correct prolyl isomers could be produced by short unfolding pulses, and its refolding kinetics could be measured. The two isomerizations in unfolded Ribonuclease T1 could be assigned to Pro39 and Pro55, because they occurred with almost identical rates in the wild-type protein, in the single-cis proline variants, and in tetrapeptide-4-nitroanilides, which contained prolines in the same sequential context at Pro39 and Pro55 or Ribonuclease T1. The direct refolding reaction of the unfolded molecules with correct prolyl isomers shows a time constant of 180 ms (at 25 degrees C, pH 4.6). This reaction is almost unaffected by the proline substitutions. It depends nonlinearly on temperature with a maximum near 25 degrees C, which suggest that the activated state for this reaction resembles the native rather than the unfolded state in heat capacity. The formation of a transient intermediate with incorrect prolyl isomers could be studied as well. Surprisingly, this reaction is only about 5-fold slower than direct folding, and it is also accompanied by a strong decrease in the apparent heat capacity.
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Folding mechanism of Ribonuclease T1 in the absence of the disulfide bonds.
Biochemistry, 1994Co-Authors: Matthias Mücke, Franz X. SchmidAbstract:In the absence of its two disulfide bonds, Ribonuclease T1 can exist in a native-like folded conformation when > or = 2 M NaCl is present. We measured the kinetics of unfolding and refolding of two reduced and carboxymethylated variants of Ribonuclease T1 with one cis proline (the Ser54Gly/Pro55Asn variant) and with two cis prolines (the wild-type protein) as a function of the NaCl concentration. Single and double mixing techniques were used. Analysis of the kinetic results demonstrates that the two cis prolyl bonds at Pro39 and Pro55 remain cis in the folded state after the reduction and carboxymethylation of the disulfide bonds. Folded molecules with trans isomers could not be found. The substitution of cis-Pro55 influences the proline-limited folding reaction, and the analysis of the changes in the folding kinetics shows that the trans-->cis isomerizations of both prolines are slow and are rate-determining steps for the refolding of Ribonuclease T1 in the presence as well as in the absence of the disulfide bonds. The direct folding reaction of protein chains with correct prolyl isomers is also affected by the Ser54Gly/Pro55Asn mutation. The rate of refolding is decreased, whereas the rate of unfolding is almost unaffected. The kinetic analysis points to two main consequences of the Ser54Gly/Pro55Asn mutation for the stability and the folding mechanism of RNase T1. It is moderately destabilizing, because the deletion of a conformationally restricted residue (Pro55-->Asn) and the insertion of a flexible residue (Ser54-->Gly) both tend to increase the entropy of the unfolded state. The cis trans isomerization of Pro55 is abolished, however, leading to a decrease in the entropy of the unfolded protein. These two entropic contributions seem to partially compensate each other, and the net change in free energy as a consequence of the Ser54Gly/Pro55Asn double mutation is very small.
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Intact disulfide bonds decelerate the folding of Ribonuclease T1
Journal of Molecular Biology, 1994Co-Authors: Matthias Mücke, Franz X. SchmidAbstract:Abstract Disulfide bonds in a folding protein chain are equivalent to prematurely formed native-like tertiary interactions. We investigated whether the mechanism of protein folding is changed by the presence of disulfide bonds. As a model we used the S54G/P55N-variant of Ribonuclease T1, a protein with two disulfide bonds and a single cis proline (Pro39), and we measured both the direct and the proline-limited folding reactions before and after breaking of the disulfide bonds. The folding kinetics were compared under refolding conditions, in the regions of the area-induced unfolding transitions of the two forms, and under folding conditions. The kinetics in the transition regions were analyzed on the basis of a three-species mechanism and all microscopic rate constants of folding and of prolyl isomerization could be determined as a function of the area concentration from the measured rates and amplitudes. These kinetic analyses indicated that the disulfide bonds can be rather unfavorable for the folding of S54G/P55N-Ribonuclease T1. Under strongly native conditions they retard the rate-limiting trans → cis isomerization of Pro39 because they allow the rapid formation of partially ordered structure prior to the proline-limited refolding reaction. Under unfolding conditions the isomerization of Pro39 is not affected. The direct unfolding and refolding reactions in the transition region of polypeptide chains with correct prolyl isomers are also decelerated when the disulfide bonds are present. These changes in the folding kinetics are possibly related to the decrease in chain flexibility that is caused by the disulfide bonds. A high flexibility is probably important throughout folding, and in the case of Ribonuclease T1 a premature locking of tertiary contacts by intact disulfide bonds can interfere unfavorably with both the direct and the proline-limited folding reactions.
Heinz Rüterjans - One of the best experts on this subject based on the ideXlab platform.
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Ionization properties of titratable groups in Ribonuclease T1. II. Electrostatic analysis.
European Biophysics Journal, 2001Co-Authors: Assen Koumanov, Heinz Rüterjans, Normann Spitzner, Andrey KarshikoffAbstract:The experimental NMR data for the individual titratable groups in Ribonuclease T1 presented in the preceding paper were analysed by means of a continuum dielectric model. The role of two factors, the alteration of hydrogen loci on the ionizable groups and the conformational flexibility, were analysed. It was suggested that the position of the titratable hydrogen is essential mainly for strongly interacting groups. For groups which are accessible to the solvent and whose ionization is not coupled with the ionization of neighbouring groups, this factor can be neglected. The influence of the conformational flexibility on the electrostatic interactions becomes apparent for the environment of K25. For some strongly interacting groups, non-sigmoidal ionization curves were calculated. On this basis the pH dependence of the NMR chemical shift of the 13Cepsilon2 resonance of H27, whose ionization is coupled with E82, was reproduced.
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Calorimetric investigation of thermal stability and ligand-binding characteristics of disulfide-bond-cleaved Ribonuclease T1.
European Journal of Biochemistry, 1995Co-Authors: Matthias F Haun, Matthias Wirth, Heinz RüterjansAbstract:A combination of differential titration calorimetry and differential scanning calorimetry was used to study the effect of disulfide bond cleavage and reaction with iodoacetamide of Ribonuclease T1 on both the binding of nucleotides and the thermal stability of the free enzyme species. Although guanosine monophosphates still bind to the active site of the modified protein the transition temperature of unfolding and the transition enthalpy decrease drastically indicating a relatively loose structure. The calorimetric data presented in this study suggest a cooperative linkage between the site of the disulfide bonds, the ligand-binding site, and the general thermodynamic stability of the enzyme.
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Backbone dynamics of Ribonuclease T1 and its complex with 2′GMP studied by two-dimensional heteronuclear NMR spectroscopy
Journal of Biomolecular NMR, 1994Co-Authors: David Fushman, Rüdiger Weisemann, Harald Thüring, Heinz RüterjansAbstract:The backbone dynamics of free Ribonuclease T1 and its complex with the competitive inhibitor 2′GMP have been studied by ^15N longitudinal and transverse relaxation experiments, combined with {^1H, ^15H} NOE measurements. The intensity decay of individual amide cross peaks in a series of (^1H, ^15N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry , 28 , 8972–8979; Kay, L.E. et al. (1992) J. Magn. Reson. , 97 , 359–375) was fitted to a single exponential by using a simplex algorithm in order to obtain ^15N T_1 and T_2 relaxation times. These experimentally obtained values were analysed in terms of the ‘model-free’ approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc. , 104 , 4546–4559; 4559–4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of Ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.
C. Nick Pace - One of the best experts on this subject based on the ideXlab platform.
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Charge–charge interactions are the primary determinants of the pK values of the ionizable groups in Ribonuclease T1 ☆
Biophysical Chemistry, 2002Co-Authors: C. Nick Pace, Gerald R. Grimsley, Beatrice M P Huyghues-despointes, James M Briggs, J Martin ScholtzAbstract:Coulomb's law and a finite difference Poisson-Boltzmann based analysis are used to predict the pK values for 15 ionizable side chains (6 Asp, 6 Glu and 3 His) in Ribonuclease T1. These predicted values are compared to the measured pK values to gain insight into the most important factors that influence the pK values of the ionizable groups in proteins. Charge-charge interactions are clearly the most important factor that determines the pK values of most ionizable groups in Ribonuclease T1. However, pK values can be shifted by several pK units by the Born self energy associated with burying ionizable groups and by favorable intramolecular hydrogen bonding.
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Charge-charge interactions are the primary determinants of the pK values of the ionizable groups in Ribonuclease T1.
Biophysical Chemistry, 2002Co-Authors: C. Nick Pace, Gerald R. Grimsley, Beatrice M P Huyghues-despointes, James M Briggs, J Martin ScholtzAbstract:Coulomb's law and a finite difference Poisson-Boltzmann based analysis are used to predict the pK values for 15 ionizable side chains (6 Asp, 6 Glu and 3 His) in Ribonuclease T1. These predicted values are compared to the measured pK values to gain insight into the most important factors that influence the pK values of the ionizable groups in proteins. Charge-charge interactions are clearly the most important factor that determines the pK values of most ionizable groups in Ribonuclease T1. However, pK values can be shifted by several pK units by the Born self energy associated with burying ionizable groups and by favorable intramolecular hydrogen bonding.
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Thermodynamics of Ribonuclease T1 denaturation.
Biochemistry, 1992Co-Authors: Julian M. Sturtevant, James Arthur Thomson, Rick E. Erickson, C. Nick PaceAbstract:Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of Ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of Ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of Ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.
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Temperature and guanidine hydrochloride dependence of the structural stability of Ribonuclease T1.
Biochemistry, 1992Co-Authors: Isabel M. Plaza Del Pino, C. Nick Pace, Ernesto FreireAbstract:The thermal unfolding of Ribonuclease T1 has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 degrees C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 degrees C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of Ribonuclease T1 as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.
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Ribonuclease T1: Structure, Function, and Stability
Angewandte Chemie International Edition in English, 1991Co-Authors: C. Nick Pace, Ulrich Hahn, Udo Heinemann, Wolfram SaengerAbstract:Proteins carry out the most important and difficult tasks in all living organisms. To do so, they must often interact specifically with other small and large molecules. This requires that they fold to a globular conformation with a unique active site that is used for the specific interaction. Consequently, protein folding can be regarded as the “secret of life”. Biochemists and chemists have a great interest in elucidating the mechanism by which proteins fold and in predicting the folded conformation and its stability given just the amino acid sequence. This challenge is sometimes called the “protein folding problem”. The ability to construct proteins differing in sequence by one or more amino acids and to analyze their three-dimensional structures by X-ray crystallography and NMR spectroscopy is a powerful tool for investigating the conformational stability and folding of proteins. Several proteins are now under intensive study by this approach. One of these is Ribonuclease T1.
