Circular dichroism studies of barnase and its mutants: characterization of the contribution of aromatic side chains.
The circular dichroism spectrum of barnase has been analyzed by examining the spectra of a series of mutants in which every single aromatic residue has been replaced. The spectrum of wild-type barnase is quite atypical for a protein of the alpha + beta class, with very low intensities and a minimum in the far-UV at 231 nm. The minimum at 231 nm is associated with the presence of Trp-94. Many other mutations involving aromatic residues have an effect on the spectral features in the far-UV. The major features in the near-UV spectra arise from essentially additive contributions of the three tryptophan residues Trp-35, Trp-71, and Trp-94. Tyrosine contributions are less prominent, with Tyr-78 and Tyr-97 contributing the most to the CD spectrum. The close charge-aromatic interaction between Trp-94 and His-18, which is important for the fluorescence properties of the protein, contributes little to the CD spectrum, as does the close aromatic-aromatic interaction between Tyr-13 and Tyr-17. However, the observed near-UV spectrum of wild-type barnase could not be simulated by the sum of the contributions of aromatic residues defined by difference spectra of protein variants carrying aromatic residues. Aromatic residues play an important role in determining the circular dichroism spectrum of proteins not only in the near-UV but also in the far-UV region.
Cambridge Centre for Protein Engineering, England, U.K.
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S
Vuilleumier
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J
Sancho
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R
Loewenthal
A R
AR
Fersht
D001426:Bacterial Proteins; D002942:Circular Dichroism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014508:Urea
D001426:Bacterial Proteins; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease