Aromatic circular dichroism in globular proteins: applications to protein structure and folding
Date
1994
Authors
Grishina, Irina Borisovna, author
Woody, Robert W., advisor
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Journal ISSN
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Abstract
The exciton couplet approach was applied to estimate the circular dichroism (CD) of Trp side-chains in proteins. Calculations were performed by the origin-independent version of the matrix method, either for the indole Bb transition only or for the six lowest energy indole transitions. The dependence of the CD of a Trp pair upon its distance and geometry has been analyzed. It was predicted that mixing with far-uv transitions are as important in determining the CD intensity of the near-uv transitions as the coupling among near-uv transition. The effects of varying exposure of Trp chromophores and nearby charges on Trp CD have been examined. A survey of a large number of proteins from the Protein Data Bank reveals a number of cases where readily detectable exciton couplets are predicted to result from the exciton coupling of Trp Bb bands. The predicted CD spectra are generally couplets, often dominated by the contributions of the closest pair, but sometimes exhibit three distinct maxima. This CD depends on the distance and relative orientation of Trp pairs and thus reflects the spatial arrangement of Trp residues in the protein. It was shown that Trp side chains can make significant contributions to the CD of proteins in the far ultraviolet. The distance dependence of exciton splitting, rotational and couplet strengths of Trp pairs show general agreement with theoretical predictions. In several cases, changes in protein Trp CD can be attributed to a specific Trp pair and explained as a definite change in its conformation. Applications of the exciton couplet approach are discussed for various crystal forms of hen lysozyme, turkey and human lysozyme. Trp62 in hen and turkey lysozymes was found to be sensitive to the perturbations of the protein surface due to binding of substrate, antibodies and intermolecular contacts in the crystal. Conformational changes of Trp62 are predicted to have a strong effect on the overall Trp CD of lysozyme. Predicted Trp CD is compared with experimental results for various lysozymes, a-chymotrypsin and chymotrypsinogen A, concanavalin, dihydrofolate reductase and ribonuclease from Bacillus intermedius 7P (binase). The calculated near-uv CD for hen lysozyme matches the experimental amplitude. Correlation of conformational changes in proteins with Trp CD is shown for a-chymotrypsin and chymotrypsinogen A. We found that the exciton couplet approach might be useful in relating Trp CD and changes in protein structure upon local mutations and conformational changes involved in enzyme activation. Small globular proteins are usually composed of a single structural domain and undergo cooperative denaturation. We have demonstrated that a protein with a single structural domain, binase, and a protein with multiple structural domains, porcine pepsin, contain fewer cooperative regions (energetic domains) under the conditions optimal for their functional activity. The study was performed by combining a CD analysis of the structural changes in the proteins during thermal denaturation and under various solvent conditions with thermodynamic properties observed by scanning microcalorimetry. Estimates of secondary structure were obtained from CD spectra, taking side-chain CD into account. It was found that neither of the proteins show any changes in secondary structure or local environment of aromatic amino acids upon separation of the energetic domains. The structural regions in binase corresponding to energetic domains were identified. It was shown that binase is converted from a single cooperative system into two separate energetic domains when ion pairs are disrupted, whereas the size of cooperative units in pepsin decrease as the electrostatic repulsion between regions in the molecule increases.
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Print version deaccessioned 2020.
Print version deaccessioned 2020.
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Subject
Circular dichroism
Globular proteins