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Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS.


ABSTRACT: Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen/high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

SUBMITTER: Pantazatos D 

PROVIDER: S-EPMC321753 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS.

Pantazatos Dennis D   Kim Jack S JS   Klock Heath E HE   Stevens Raymond C RC   Wilson Ian A IA   Lesley Scott A SA   Woods Virgil L VL  

Proceedings of the National Academy of Sciences of the United States of America 20040108 3


Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen/high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization suc  ...[more]

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