A comparative CD study of carbonic anhydrase isoenzymes with different number of tryptophans: Impact on calculation of secondary structure content

Abstract

The CD spectra of human carbonic anhydrase I and I1 and bovine carbonic anhydrase 111 were recorded and analyzed. The 3D structures of these isoenzymes are known, showing very similar secondary structure and polypeptide-chain fold. The tryptophan content, however, differs between the isoenzymes, i.e., isoenzymes I, 11, and 111 possess 6, 7, and 8 tryptophans, respectively. All of the tryptophans except the additional tryptophans in isoenzymes I1 and 111, i.e., W245 and W47, are conserved. Despite the fact that X-ray structure determinations showed that the isoenzymes had highly similar secondary structure, the contents of a-helix and p-sheet structure differed considerably when using different CD algorithms for estimation of the fractions of various secondary structural elements. This shows that aromatic amino acids also interfere in the wavelength region (far-UV) used to calculate the amount of secondary structure. Such interference is especially problematic when analyzing proteins like carbonic anhydrase, which consist mainly of p-structure that gives rise to weak ellipticity bands, compared to the bands arising from a-helical structure.Keywords: aromatic amino acids; bovine carbonic anhydrase 111; p-structure; human carbonic anhydrase I and I1Proteins exhibit characteristic CD spectra in the far-UV region and the appearance of these spectra are related to the presence of secondary structure (Greenfield & Fasman, 1969 Compton & Johnson, 1986;Manavalan &Johnson, 1987;Perczel et al., 1991). Knowledge of the secondary structure is of great importance in the understanding of the function, stability, and folding of a protein, and CD estimations of the amount of a-helices have been particularly accurate for many proteins. On the other hand, it has been more difficult to predict the contents of p-structure (Sreerama & Woody, 1994). One obvious reason for this is that the a-helix spectrum dominates the protein far-UV CD spectrum (Johnson, 1990;Sreerama & Woody, 1993), and the CD signal arising from p-structure is of lower intensity. This means that calculations of the amount of this type of secondary structure are more sensitive to perturbations from other contributing structural elements. Based on theoretical calculations and experimental evidence from peptide models, it has been suggested that aromatic amino acids make contributions to the far-UV CD spectrum (Strickland, 1974; ManReprint ning & Woody, 1989;Manning et al., 1992). Because this wavelength region is used. for estimation of the amount of secondary structure and the models used do not consider aromatic contributions explicitly, aromatic amino acids could seriously interfere with such calculations. In a recent study, we used site-directed mutagenesis to replace one Trp at a time in human carbonic anhydrase I1 (HCA 11) in order to assign the contribution of each of the Trp residues to the CD spectrum (Freskgkd et al., 1994). We found that the individual Trp residues contributed to the CD spectrum not only in the near-UV wavelength region, but also...

Thermodynamics of Metal Ion Binding. 2. Metal Ion Binding by Carbonic Anhydrase Variants

“…c Calculated from KD values at 25°C. characteristic bands of T199A CAII are all present in CD spectra reported for WT CAII (31). The CD spectra of the two proteins are essentially identical, with the apo-T199A CAII spectrum showing no inconsistencies from the holoenzyme spectrum that might indicate a mixture of differently folded enzyme.…”

The Structure of Carbonic Anhydrase IX Is Adapted for Low-pH Catalysis

“…As a reference standard, we note that the spectra observed for the hCA II pH 7.0 sample are consistent with previously collected CD data for the enzyme. 51 The difference in the spectra for hCA II at pH 3.0 indicates possible changes in β -sheet content after incubation in an acidic environment as the negative peak changes from 210 nm at pH 7.0 to 202 nm at pH 3.0. This further correlates to DSF and DSC data that suggest a complete denaturation of hCA II at pH <5.0.…”

Folding mechanism of three structurally similar β-sheet proteins

“…Both CRABP I and IFABP showed a small negative signal over this wavelength range, but no signal was detected for CRBP II. These intensity differences were probably due to the contribution of aromatic residues to the far-UV CD signal, as shown for carbonic anhydrase 40 and barnase. 41 Mutations of W87 and W109 (W87F, W109F) of CRABP I altered the CD signal around 228 nm, suggesting that these residues are involved in the CD spectral differences.…”