Determining the Conformational Changes within an Active Enzyme-Substrate System on both Sides of the Reaction.
EMSL Project ID
47443
Abstract
Our objective is to develop solution-based strategies that directly monitor the physical conformational changes that occur within enzymes, both free and during enzyme catalysis. These strategies are reliant on multiple advances in both nuclear magnetic resonance (NMR) spectroscopy and computational techniques, which will be combined here to reveal the movements that occur in an active complex as well determine how movements in the free enzyme are inherently poised for function. A major, but yet essentially unexploited advantage of NMR spectroscopy is that this technique can be used to monitor active enzymes in solution in order to obtain the atomic resolution details of catalysis. Importantly, our applications of NMR methods to enzymes in both the absence and presence of substrates has allowed us to begin to determine how the inherent flexibility (i.e. dynamics) is related to function by monitoring their dynamics. While these methods have proven invaluable in identifying regions of proteins that exhibit dynamics, they have not revealed the actual physical movements that are associated with the dynamics measurements. A complete understanding of the conformational changes associated with the dynamics measurements would have far-reaching applications. For example, directly probing the conformational changes of active enzymes would permit us to directly determine mechanisms and may then allow us to rationally engineer movements in order to control catalytic function. The potential of dynamically designing biocatalysts would have far-reaching applications to many fields that include biomedical and environmental sciences. Thus, we now propose to apply recently developed structure-based methods to an active enzyme to reveal the associated physical conformational changes that our dynamics studies have measured.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2012-10-01
End Date
2014-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Camilloni C, AB Sahakyan, M Holliday, NG Isern, F Zhang, EZ Eisenmesser, and M Vendruscolo. 2014. "Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism." Proceedings of the National Academy of Sciences of the United States of America 111(25):, doi:10.1073/pnas.1404220111
Doshi U ,Holliday M ,Eisenmesser E Z,Hamelberg D 2016. "Dynamical Network of Residue–Residue Contacts Reveals Coupled Allosteric Effects in Recognition, Catalysis, and Mutation" Proceedings of the National Academy of Sciences of the United States of America 113(17):4735–4740. 10.1073/pnas.1523573113
Eisenmesser EZ, G Capodagli, G Armstrong, MJ Holliday, NG Isern, F Zhang, and SD Pegan. 2015. "Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions." Protein Science. doi:10.1002/pro.2637
Holliday M, F Zhang, NG Isern, GS Armstrong, and EZ Eisenmesser. 2014. "1H, 13C, and 15N backbone and side chain resonance assignments of thermophilic Geobacillus kaustophilus cyclophilin-A." Biomolecular NMR Assignments 8(1):23-27. doi:10.1007/s12104-012-9445-3
Kendrick A, M Holliday, NG Isern, F Zhang, C Camilloni, C Huynh, M Vendruscolo, GS Armstrong, and EZ Eisenmesser. 2014. "The dynamics of interleukin-8 and its interaction with human CXC receptor I peptide." Protein Science 23(4):464-480. doi:10.1002/pro.2430