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Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

Lookup NU author(s): Dr Chan Li, Sachiko Yanagisawa, Dr Mark Banfield, Professor Christopher Dennison

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Abstract

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C112TFPGH117SALM 121 to C112TPH115PFM118 (the native loop from the cupredoxin amicyanin) and also to C112TPH 115PM117. The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the C112TPH115PM117 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the C112TPH115PFM118 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein. © 2006 by The National Academy of Sciences of the USA.


Publication metadata

Author(s): Li C, Yanagisawa S, Martins BM, Messerschmidt A, Banfield MJ, Dennison C

Publication type: Article

Publication status: Published

Journal: Proceedings of the National Academy of Sciences of the United States of America

Year: 2006

Volume: 103

Issue: 19

Pages: 7258-7263

ISSN (print): 0027-8424

ISSN (electronic): 1091-6490

Publisher: National Academy of Sciences

URL: http://dx.doi.org/10.1073/pnas.0600774103

DOI: 10.1073/pnas.0600774103

PubMed id: 16651527


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