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Formation of Local Native-like Tertiary Structures in the Slow Refolding Reaction of Human Carbonic Anhydrase II as Monitored by Circular Dichroism on Tryptophan Mutants

Andersson, Dick and Freskgard, Per-Ola and Jonsson, Bengt-Harald and Carlsson, Uno (1997) Formation of Local Native-like Tertiary Structures in the Slow Refolding Reaction of Human Carbonic Anhydrase II as Monitored by Circular Dichroism on Tryptophan Mutants. Biochemistry, 36 (15). pp. 4623-4630. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20180212-133230052

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Abstract

In the present study, near-UV CD kinetic measurements on mutants, in which one Trp residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCAII). In addition, the formation of the active site was probed by the binding of a fluorescent sulfonamide inhibitor. The development of the individual Trp CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCAII at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Trp residues 16, 97, and 245 were analyzed. Trp16 probes the N-terminal domain (amino acid residues 1−25), and this part is forming its tertiary structure slower than the major domain (amino acid residues 26−260) of the protein molecule, which contains the active site and a dominating β-sheet. An essentially native structure of the major domain seems to act as a template for the correct folding of the N terminus. Trp97 is located in a hydrophobic cluster comprising β-strands 3−5 in the protein core. Previously, we have shown that this region is remarkably stable and compact, and stopped-flow fluorescence data indicate that Trp97 is buried in an apolar compact cluster within a few milliseconds [Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Mårtensson, L.-G., Gentile, M., Borén, K., & Carlsson, U. (1995) Biochemistry 34, 8606−8620; Jonasson, P., Aronsson, G., Carlsson, U., & Jonsson, B.-H. (1997) Biochemistry 36 (in press)]. Here it is shown that the development of the native tertiary structure at Trp97 occurs in the minute time domain. Trp245 is located in a long loop between the N-terminal domain and the core structure. Although this Trp has attained native-like fluorescence properties within the dead time of the CD experiment, it assumes a native-like asymmetric environment even slower than Trp97. Thus, the investigated Trp residues develop their native CD bands at different rates, showing that formation of native-like tertiary structure is occurring with varying rates in different regions of the protein.


Item Type:Article
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http://dx.doi.org/10.1021/bi961925nDOIArticle
https://pubs.acs.org/doi/10.1021/bi961925nPublisherArticle
Additional Information:© 1997 American Chemical Society. Received 5 August 1996. Published online 15 April 1997. This work was supported by grants from the Swedish National Board for Industrial and Technical Development (88-04439P, U.C.; 88-04439P, B.-H.J.), the Swedish Natural Science Research Council (K-Ku 4241-301, U.C.; K-Ku 9426-300, B.-H.J.), the Sven and Lily Lawskis Fond (P.-O.F.), and the Marcus and Amalia Wallenbergs Minnesfond (U.C.). We thank Göran Aronsson (Umeå University) for making Figure 1.
Funders:
Funding AgencyGrant Number
Swedish National Board for Industrial and Technical Development88-04439P
Swedish Natural Science Research CouncilK-Ku 4241-301
Swedish Natural Science Research CouncilK-Ku 9426-300
Sven and Lily Lawskis FondUNSPECIFIED
Marcus and Amalia Wallenbergs MinnesfondUNSPECIFIED
Issue or Number:15
Record Number:CaltechAUTHORS:20180212-133230052
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180212-133230052
Official Citation:Formation of Local Native-like Tertiary Structures in the Slow Refolding Reaction of Human Carbonic Anhydrase II as Monitored by Circular Dichroism on Tryptophan Mutants Dick Andersson, Per-Ola Freskgård, Bengt-Harald Jonsson, and Uno Carlsson Biochemistry 1997 36 (15), 4623-4630 DOI: 10.1021/bi961925n
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84792
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:13 Feb 2018 21:12
Last Modified:03 Oct 2019 19:22

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