Utilizing H/D-Exchange to Identify Biological Structures
• Identification of disordered regions in protein constructs that prevent crystallization
• Identification of optimum crystallization conditions
• Identifying the effect of point mutations on protein stability (e.g., wild-type protein vs. congenital mutations)
Proteins flex, unfold, refold and possess a variety of motions important to their function. Amide hydrogen/deuterium exchange, coupled with proteolysis and liquid chromatography-mass spectrometry (H/D-Ex) is becoming a popular method for probing these motions¹. For example, large collaborative protein structure initiatives, such as the NESG and JCSG, regularly employ H/D-Ex to help design well-folded, highly stable protein constructs. This is due to the ability of H/D-Ex to reliably delineate automonous structural domains from disordered regions2-4.
1. Hamuro Y, Coales SJ, Southern MR, Nemeth-Cawley JF, Stranz DD, Griffin PR. Rapid analysis of protein structure and dynamics by hydrogen/deuterium exchange mass spectrometry. J Biomol Tech. 2003;14:171-182.
2. Sharma S, Zheng H, Huang YJ, Ertekin A, Hamuro Y, Rossi P, et al. Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry. Proteins. 2009;76(4):882-894.
3. Pantazatos D, Kim JS, Klock HE, Stevens RC, Wilson IA, Lesley SA, et al. Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS. P Natl Acad Sci USA. 2004;101(3):751-756.
4. Spraggon G, Pantazatos D, Klock HE, Wilson IA, Woods VL Jr., Lesley SA. On the use of DXMS to produce more crystallizable proteins: structures of the T. maritima proteins TM0160 and TM1171. Protein Sci. 2004;13:3187-3199.