Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the1La and1Lb component spectra

Barth, Andreas; Martin, Stephen R.; Bayley, Peter M.

doi:10.1002/(sici)1097-0282(199806)45:7<493::aid-bip3>3.0.co;2-j

Cited by 21 publications

(18 citation statements)

References 23 publications

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“…The near-UV of SmelDhp was very intense: it has a broad minimum centered at 280 nm, and another shallow one at 290 nm (Fig. 4 C, filled squares), suggesting a very intense L b transition of the tryptophans [33,34]. In the presence of the ion, the spectrum is more intense (Fig.…”

Section: (D) Far and Near-uvmentioning

confidence: 99%

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Martínez‐Rodríguez

Encinar

Hurtado-Gómez

et al. 2009

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 3 ABSTRACTDihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-β-amino acids. This enzyme is an attractive candidate for commercial production of D-amino acids, which are used in the production of semisynthetic β-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH ~6.0, which is stable up to pH ~9.0, as concluded from fluorescence and CD.Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.

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Section: (D) Far and Near-uvmentioning

confidence: 99%

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Martínez‐Rodríguez

Encinar

Hurtado-Gómez

et al. 2009

Biophysical Chemistry

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“…The resolutions of the NATA spectra into the 1 L a and 1 L b components were achieved by using the fitting strategy described in detail elsewhere [15]. Briefly, the 1 L b band of Barth et al [14] was deconvolved into five Gaussian components. The existence of the five vibronic bands has been shown experimentally for the 1 L b of a related model compound, 5-methoxyindole [25].…”

Section: Fitting Of the Absorption And CD Spectramentioning

confidence: 99%

“…The line widths and amplitudes of the Gaussian components were varied to improve fitting of the 1 L b band in various solvents. However, no deconvolution was necessary for the 1 L a band of Barth et al [14]. As a result, the set of the 1 L b components, each of which corresponds to NATA in a particular solvent, was obtained.…”

Section: Fitting Of the Absorption And CD Spectramentioning

confidence: 99%

“…Some success has been achieved in resolving the near-UV CD spectra of Trp by varying the relative intensity of the 0-0 transition of 1 L b in the fitting procedure. Relative intensities of vibronic bands of 1 L b vary for Trp in buffer compared with that included in a protein structure [14]. The 1 L b transition, in contrast to the 1 L a , is quite sensitive to the methyl or methoxy substitutions on the indole ring, particularly at positions 4, 6, and 7.…”

Section: Introductionmentioning

confidence: 96%

“…Previously, a few attempts have been made to resolve the near-UV CD spectra of Trp by fitting the spectra to the sum of the 1 L a and 1 L b components [14,15]. It has been shown that one type of the 1 L b spectrum is not sufficient for the fitting of certain types of tryptopan CD spectra.…”

Section: Introductionmentioning

confidence: 99%

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Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Gasymov

Abduragimov

Glasgow

2008

Analytical Biochemistry

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The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1 L a and 1 L b components. The relative intensities of the 0-0 transitions of the 1 L b bands correlate linearly with the solvent polarity values . A novel strategy, which utilizes a set of the experimental 1 L b bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1 L b bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The "DMSOlike" 1 L b band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data.

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Structure and conformational stability of a tetrameric thermostableN‐succinylamino acid racemase

et al. 2009

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Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the1La and1Lb component spectra

Cited by 21 publications

References 23 publications

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

Structure and conformational stability of a tetrameric thermostableN‐succinylamino acid racemase

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