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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM101135-04
Application #
8928002
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2012-09-30
Project End
2016-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
4
Fiscal Year
2015
Total Cost
$295,450
Indirect Cost
$105,450
Name
Northeastern University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001423631
City
Boston
State
MA
Country
United States
Zip Code
02115
Wilson, Christopher J; Das, Madhurima; Jayaraman, Shobini et al. (2018) Effects of Disease-Causing Mutations on the Conformation of Human Apolipoprotein A-I in Model Lipoproteins. Biochemistry 57:4583-4596
de Wispelaere, Melissanne; Lian, Wenlong; Potisopon, Supanee et al. (2018) Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein. Cell Chem Biol 25:1006-1016.e8
Moroco, Jamie A; Alvarado, John Jeff; Staudt, Ryan P et al. (2018) Remodeling of HIV-1 Nef Structure by Src-Family Kinase Binding. J Mol Biol 430:310-321
Kochert, Brent A; Iacob, Roxana E; Wales, Thomas E et al. (2018) Hydrogen-Deuterium Exchange Mass Spectrometry to Study Protein Complexes. Methods Mol Biol 1764:153-171
Pritz, Jonathan R; Wachter, Franziska; Lee, Susan et al. (2017) Allosteric sensitization of proapoptotic BAX. Nat Chem Biol 13:961-967
Wales, Thomas E; Fadgen, Keith E; Eggertson, Michael J et al. (2017) Subzero Celsius separations in three-zone temperature controlled hydrogen deuterium exchange mass spectrometry. J Chromatogr A 1523:275-282
Lu, Jia; Harrison, Rane A; Li, Lianbo et al. (2017) KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry. Structure 25:1442-1448.e3
Das, Madhurima; Wilson, Christopher J; Mei, Xiaohu et al. (2017) Structural stability and local dynamics in disease-causing mutants of human apolipoprotein a-I: what makes the protein amyloidogenic? Amyloid 24:11-12
Harrison, Rane A; Engen, John R (2016) Conformational insight into multi-protein signaling assemblies by hydrogen-deuterium exchange mass spectrometry. Curr Opin Struct Biol 41:187-193
Das, Madhurima; Wilson, Christopher J; Mei, Xiaohu et al. (2016) Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic? J Mol Biol 428:449-62

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