Assignment of the contribution of the tryptophan residues to the circular dichroism spectrum of human carbonic anhydrase II

Abstract

The circular dichroism (CD) spectrum of human carbonic anhydrase II (HCAII) has been investigated using various mutants of the enzyme in which tryptophans have been replaced by site-directed mutagenesis. HCAII contains seven tryptophans which are believed to significantly contribute to the CD spectrum in both the near- and far-UV regions. By substituting the tryptophans one at a time, the spectral effects of the individual tryptophans were studied. The near-UV spectrum of HCAII is very complex, with multiple Cotton effects. This complexity has been attributed to aromatic amino acids, especially tryptophans, located in asymmetric aromatic clusters in the molecule. CD spectra of the individual tryptophans were calculated as difference spectra between the CD spectrum of HCAII and those of the tryptophan mutants. These spectra showed that the tryptophans contributed to the CD spectrum in almost the entire wavelength region investigated (180-310 nm). Summation of the individual tryptophan CD spectra in the near-UV region yielded a spectrum that was qualitatively very similar to that of HCAII, showing that the tryptophans are the major determinant for this part of the CD spectrum. Since tryptophans were also demonstrated to contribute significantly in the far-UV region, tryptophans can interfere considerably with the assignment of changes in CD bands to changes in secondary structure content during folding reactions. Moreover, because of this substantial interference, predictions of the amount of various types of secondary structure from CD data from the far-UV region are made more difficult. These findings are probably of general importance for proteins that, like HCAII, contain several tryptophans.(ABSTRACT TRUNCATED AT 250 WORDS)