Designing Sequence to Control Protein Function in an EF-Hand Protein
The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca2+ sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca2+ binding, comparative analyses o...
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Published in | Journal of the American Chemical Society Vol. 126; no. 19; pp. 5990 - 5998 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Washington, DC
American Chemical Society
19.05.2004
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Subjects | |
Online Access | Get full text |
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Summary: | The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca2+ sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca2+ binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca2+-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D9k re-engineered with 15 mutations to respond to Ca2+ binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca2+-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca2+ sensorlike conformational changes. The X-ray crystal structure of Ca2+-CBM-1 determined at 1.44 Å resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design. |
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Bibliography: | istex:91B2047106C843AD02A23A9A84916F990486FCA4 ark:/67375/TPS-MVB5QMJ2-J ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0002-7863 1520-5126 |
DOI: | 10.1021/ja0397456 |