To perform their biological function, individual proteins associate, often in a transient manner, to form complexes. Understanding the way complexes function is a far-reaching scientific goal for disciplines ranging from molecular medicine to physical chemistry. While high- detail structural information can sometimes be obtained by X-ray diffraction analysis, this requires the availability of a sufficient quantity of homogenous material and definition of suitable crystallization parameters. Both conditions are often difficult to meet for large complexes and for membrane proteins and thus the number of these structures deposited in databases remains relatively low. Alternative methodologies such as electron microscopy (EM) and small angle X- ray scattering (SAXS) allow determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited, in general, to homogeneous complexes. Consequently there is a need to develop new approaches that define subunit stoichiometry, composition, shape, and the dynamics of heterogeneous macromolecular complexes of biomedical importance particularly those in intact biological membranes and organelles. This proposal combines new crosslinker strategies and ion mobility (IM) coupled to mass spectrometry (MS) jointly as high-throughput structural probes for multi-protein complexes, particularly membrane complexes. The development of new crosslinker strategies based on small molecule chemistries consistent with the requirements of MS and IM will overcome many of the existing constraints for analysis of protein complexes. This is a first step towards developing a suite of new high-throughput mass spectrometry-based technologies that will enable the discovery of many previously-unknown multi-protein complex structures and will provide peptide proximity information of use for interpreting structures and providing constraints for protein folding calculations. Importantly, it will also provide a basis for developing methods for following interaction dynamics in protein complexes.

Public Health Relevance

Membrane proteins represent attractive drug targets but their unique physical properties make their structures difficult to determine and only a small fraction have had their structures determined with sufficient accuracy to be useful which limits opportunities for rational drug design. This proposal will develop high-throughput chemical and physical technologies to determine the membrane topologies of proteins and the interactions between membrane proteins that lead to function. These technologies will provide structural constraints useful in refining 3-D topology diagrams of multi-protein complexes, in de novo protein folding, defining protein-protein interactions and to study the dynamics of protein complexes in normal and diseased tissues to identify the specific protein complexes perturbed in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM095832-02
Application #
8249811
Study Section
Special Emphasis Panel (ZRG1-BST-N (50))
Program Officer
Edmonds, Charles G
Project Start
2011-04-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$307,876
Indirect Cost
$101,418
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Dixit, Sugyan M; Polasky, Daniel A; Ruotolo, Brandon T (2018) Collision induced unfolding of isolated proteins in the gas phase: past, present, and future. Curr Opin Chem Biol 42:93-100
Avtonomov, Dmitry M; Polasky, Daniel A; Ruotolo, Brandon T et al. (2018) IMTBX and Grppr: Software for Top-Down Proteomics Utilizing Ion Mobility-Mass Spectrometry. Anal Chem 90:2369-2375
Polasky, Daniel A; Lermyte, Frederik; Nshanian, Michael et al. (2018) Fixed-Charge Trimethyl Pyrilium Modification for Enabling Enhanced Top-Down Mass Spectrometry Sequencing of Intact Protein Complexes. Anal Chem 90:2756-2764
Eschweiler, Joseph D; Farrugia, Mark A; Dixit, Sugyan M et al. (2018) A Structural Model of the Urease Activation Complex Derived from Ion Mobility-Mass Spectrometry and Integrative Modeling. Structure 26:599-606.e3
Hagen, Susan E; Liu, Kun; Jin, Yafei et al. (2018) Synthesis of CID-cleavable protein crosslinking agents containing quaternary amines for structural mass spectrometry. Org Biomol Chem 16:8245-8248
Niu, Shuai; Kim, Byung Chul; Fierke, Carol A et al. (2017) Ion Mobility-Mass Spectrometry Reveals Evidence of Specific Complex Formation between Human Histone Deacetylase 8 and Poly-r(C)-binding Protein 1. Int J Mass Spectrom 420:9-15
Soper-Hopper, Molly T; Eschweiler, Joseph D; Ruotolo, Brandon T (2017) Ion Mobility-Mass Spectrometry Reveals a Dipeptide That Acts as a Molecular Chaperone for Amyloid ?. ACS Chem Biol 12:1113-1120
Eschweiler, Joseph D; Frank, Aaron T; Ruotolo, Brandon T (2017) Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment. J Am Soc Mass Spectrom 28:1991-2000
Haynes, Sarah E; Polasky, Daniel A; Dixit, Sugyan M et al. (2017) Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics. Anal Chem 89:5669-5672
Eschweiler, Joseph D; Martini, Rachel M; Ruotolo, Brandon T (2017) Chemical Probes and Engineered Constructs Reveal a Detailed Unfolding Mechanism for a Solvent-Free Multidomain Protein. J Am Chem Soc 139:534-540

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