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Increased Backbone Flexibility in Threonine45-Phosphorylated Hirudin upon pH Change

Protein phosphorylation on serine/threonine side chains represents a major regulatory event in the posttranslational control of protein functionality, where it is thought to operate at the level of structural changes in the polypeptide chain. However, key questions about molecular aspects of phospha...

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Bibliographic Details
Published in:Biochemistry (Easton) 2001-07, Vol.40 (27), p.7957-7963
Main Authors: Kipping, Marc, Zarnt, Toralf, Kiessig, Steffen, Reimer, Ulf, Fischer, Gunter, Bayer, Peter
Format: Article
Language:English
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Summary:Protein phosphorylation on serine/threonine side chains represents a major regulatory event in the posttranslational control of protein functionality, where it is thought to operate at the level of structural changes in the polypeptide chain. However, key questions about molecular aspects of phosphate ester induced conformational alterations remain open. Among these concerns are the radius of action of the phosphate ester group, its effective ionic state, and its interplay with distinct bonds of the polypeptide chain. Primarily to define short-range effects upon threonine phosphorylation, the native 65 amino acid protein hirudin, conformationally restrained by a proline flanking the pThr45 site and three intramolecular disulfide bonds, was structurally characterized in both the phosphorylated and the unphosphorylated state in solution. Circular dichroism and hydrogen exchange experiments (MALDI-TOF) showed that structural changes were caused by Thr45-Pro46 phosphorylation only when the phosphate ester group was in its dianionic state. The spatial arrangement of the amino acids, monitored by 1H NMR spectroscopy, appears to be affected within a radius of about 10 Å around the pThr45-OγΗ, with phosphorylation resulting in a loss of structure and increased flexibility within a segment of at least seven amino acid residues. Thus, the transition from the monoanionic to the dianionic phosphate group over the pH range 5.2−8.5 represents a general phosphorylation-dependent conformational switch operating at physiological pH values.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi010317r