Denaturation studies by carbon-13 nuclear magnetic resonance on modified basic pancreatic trypsin inhibitor using the novel S-[13C]methyl methionyl probe

Harina, Benjamin M.; Dyckes, Douglas F.; Willcott, M. Robert; Jones, Warren C. Jun.

doi:10.1021/ja00523a031

Cited by 11 publications

(3 citation statements)

References 7 publications

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“…as well as the overall contribution of each site to the total electrostatic free energy of the molecule (Friend & Gurd, 1979a,b;Friend et al, 1980Friend et al, , 1981Flanagan et al, 1981). Because of the exceptional stability of the bovine pancreatic trypsin inhibitor (BPTI) to extremes of pH and temperature (Vincent et al, 1971; Masson & Wüthrich, 1973;Harina et al, 1980; as well as its small size (Huber et al, 1970;Deisenhofer & Steigemann, 1975), it presents an opportunity to test the prediction of pK values for acidic and basic residues. The titrations of various amino acid residues in BPTI have been reported under many different conditions of ionic strength and temperature (Maurer et al, 1974;Snyder et al, 1975;Wagner & Wüthrich, 1975;Brown et al, 1976Brown et al, , 1978 & , making comparisons with predictions of the electrostatic theory difficult.…”

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confidence: 99%

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

March¹,

Maskalick²,

England³

et al. 1982

Biochemistry

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The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.

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confidence: 99%

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

March¹,

Maskalick²,

England³

et al. 1982

Biochemistry

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“…NMR relaxation techniques can be used to detect and quantify picosecond motions but are often of limited value due to the low natural abundance of the isotope which necessitates the use of high and frequently unattainable protein concentrations. With selective enrichment the method becomes particularly appealing and measurement of NMR relaxation parameters has clearly demonstrated the occurence of very rapid motions in proteins and peptides (Niu et al, 1979;Eakin et al, 1975;Jones et al, 1976;Blakeley et al, 1978;Deber et al, 1978;Cohen et al, 1979;Harina et al, 1980;Matta et al, 1980;Wooten et al, 1981;Hughes et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy

Weaver¹,

Kemple²,

Prendergast³

1988

Biophysical Journal

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Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter. In lysolecithin micelles, the model-free approach applied tot-boc-LALALW-OMe predicts a Te between 0.87 and 1.08 ns, and an order parameter range of 0.72-0.80, assuming an average Tm of 14 ns (Saunders, L. 1966. Biochim. Biophys. Acta. 125:70) for a typical peptide-micelle complex. In this case, measurement of only two 13C relaxation parameters at a single frequency yields sufficient information, plotted in the form of a composite T1-NOE solution curve, to constrain the allowed values of the model-free motional parameters within a relatively narrow range. The predicted range of eV and Te values for the pepti...

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“…published (Eakin et al, 1975;Jones et al, 1976;Blakley et al, 1978;Deber et al, 1978;Schejter et al, 1978;Harina et al, 1980;Matta et al, 1980;Wooten et al, 1981). These have frequently been prepared biosynthetically, resulting in the presence of multiple peaks in the spectrum and the need for subsequent assignment.…”

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confidence: 99%

Carbon-13 NMR studies of the molecular dynamics of selectively carbon-13-enriched ribonuclease complexes

Hughes¹,

Cohen²,

Szabó³

et al. 1984

Biochemistry

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13C spin-lattice (T1) relaxation times determined at four frequencies (25, 68, 100, and 125 MHz) have been used to probe the molecular dynamics of ribonuclease S' complexes prepared from synthetic amino-terminal peptides containing 13C enrichment (ca. 90%) at selected sites [Niu, C., Matsuura, S., Shindo, H., & Cohen, J. S. (1979) J. Biol. Chem. 254, 3788]. It was found that the motion of the C alpha-H bond of Ala-5 could not be determined by isotropic reorientation alone. The time scale and spatial restriction on the internal motion of this residue were determined by the model-free approach of Lipari and Sazbo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. It was found that the C alpha-H bond, in addition to an overall correlation time of 20 ns, underwent internal motion with a correlation time of 0.5 ns and a generalized order parameter S corresponding to a cone semiangle of 23 degrees C. The C beta-H bond had a correlation time of 37 ps, reflecting the fast rotation of the methyl group, and had an S value close to that expected if the C alpha-C beta and C alpha-H bonds have the same degree of spatial restriction.

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Denaturation studies by carbon-13 nuclear magnetic resonance on modified basic pancreatic trypsin inhibitor using the novel S-[13C]methyl methionyl probe

Cited by 11 publications

References 7 publications

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy

Carbon-13 NMR studies of the molecular dynamics of selectively carbon-13-enriched ribonuclease complexes

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