There are a great number of proteins and protein complexes whose function depends on structural flexibility and conformational changes that remain uncharted and unknown and well beyond the reach of current analytical methodologies. Many of these protein systems are critical components of essential biochemical pathways, contributors - both positive and negative - to disease processes, and relevant to a broad range of human health-related problems. Progress in understanding these systems will depend on development of new tools capable of studying the structural basis of function in these proteins. Hydrogen exchange (HX) mass spectrometry (MS) can be used for studies of conformation and flexibility of many proteins not suitable for analysis with conventional tools. But for those systems of greatest current interest - including protein complexes, membrane proteins, and large (>250 kDa) systems - existing HX MS techniques are inadequate. The objective of this project is to develop, refine and reduce to common practice HX MS methods that overcome the current limitations of the approach. We will do this by completing three specific aims: (1): Combine HX MS and affinity capture techniques to enhance the amount of information that can be obtained in the study of protein complexes through HX MS;(2): Extend conformational studies to membrane proteins by adapting the use of nanodiscs as appropriate vehicles for membrane proteins studied by HX MS;(3): Integrate ion mobility spectrometry (IMS) into the HX MS workflow to enhance the resolving power and thereby substantially increase the upper limit of protein size to which HX MS can be applied. We will use innovative experimental design to make possible directed isolation of specific proteins from complexes while maintaining the restrictive quench conditions required by HX MS methodology. Nanodiscs will be used to provide relevant membrane environments for the study of conformation and flexibility of membrane proteins. The expected outcome will be a roadmap for the routine use of HX MS for conformational analysis of large protein complexes and membrane proteins, demonstrated through the detailed conformational analysis of several disease- relevant test protein systems. These test systems include complexes important for HIV progression, cellular signaling in leukemia, membrane-associated proteins important for pain, inflammation and neurodegenerative disorders, and a transmembrane protein important in vitamin-K dependent blood coagulation. The proposed research is significant because it is expected to advance the field of protein conformational analysis with mass spectrometry, making possible analysis of the conformation and dynamics of proteins and protein systems for which conformational information cannot otherwise be obtained. These tools would then be applied by ourselves and in coordination with others to study a huge variety of proteins, protein complexes, and problems in the fields of protein science and medicine.
The proposed research, development of methods that allow for the analysis of the conformation of proteins not amenable to other techniques, is relevant for public health because understanding protein structure is essential to understanding abnormal, disease-related protein function. The results will provide tools relevant to NIH's mission to develop new bioanalytical methods in order to lay the foundation for advances in disease diagnosis, treatment and prevention.
|Boyken, Scott E; Chopra, Nikita; Xie, Qian et al. (2014) A conserved isoleucine maintains the inactive state of Bruton's tyrosine kinase. J Mol Biol 426:3656-69|
|Wei, Hui; Mo, Jingjie; Tao, Li et al. (2014) Hydrogen/deuterium exchange mass spectrometry for probing higher order structure of protein therapeutics: methodology and applications. Drug Discov Today 19:95-102|
|Iacob, Roxana E; Chen, Guodong; Ahn, Joomi et al. (2014) The influence of adnectin binding on the extracellular domain of epidermal growth factor receptor. J Am Soc Mass Spectrom 25:2093-102|
|Fang, Jing; Nevin, Philip; Kairys, Visvaldas et al. (2014) Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics. Structure 22:572-81|
|Lim, Sang Min; Westover, Kenneth D; Ficarro, Scott B et al. (2014) Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor. Angew Chem Int Ed Engl 53:199-204|
|Parker, Christine H; Morgan, Christopher R; Rand, Kasper D et al. (2014) A conformational investigation of propeptide binding to the integral membrane protein ?-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53:1511-20|
|Georgescauld, Florian; Popova, Kristina; Gupta, Amit J et al. (2014) GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel domain folding. Cell 157:922-34|
|Wang, Weixue; Iacob, Roxana E; Luoh, Rebecca P et al. (2014) Electron transfer control in soluble methane monooxygenase. J Am Chem Soc 136:9754-62|
|Karageorgos, Ioannis; Wales, Thomas E; Janero, David R et al. (2013) Active-site inhibitors modulate the dynamic properties of human monoacylglycerol lipase: a hydrogen exchange mass spectrometry study. Biochemistry 52:5016-26|
|Tsukamoto, Kosuke; Wales, Thomas E; Daniels, Kathleen et al. (2013) Noncanonical role of the PDZ4 domain of the adaptor protein PDZK1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class B, type I (SR-BI). J Biol Chem 288:19845-60|
Showing the most recent 10 out of 21 publications