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.
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.
|Samulak, Billy M; Niu, Shuai; Andrews, Philip C et al. (2016) Ion Mobility-Mass Spectrometry Analysis of Cross-Linked Intact Multiprotein Complexes: Enhanced Gas-Phase Stabilities and Altered Dissociation Pathways. Anal Chem 88:5290-8|
|Han, Linjie; Ruotolo, Brandon T (2015) Ion Mobility-Mass Spectrometry Differentiates Protein Quaternary Structures Formed in Solution and in Electrospray Droplets. Anal Chem 87:6808-13|
|Farrugia, Mark A; Han, Linjie; Zhong, Yueyang et al. (2013) Analysis of a soluble (UreD:UreF:UreG)2 accessory protein complex and its interactions with Klebsiella aerogenes urease by mass spectrometry. J Am Soc Mass Spectrom 24:1328-37|
|Soper, Molly T; DeToma, Alaina S; Hyung, Suk-Joon et al. (2013) Amyloid-Î²-neuropeptide interactions assessed by ion mobility-mass spectrometry. Phys Chem Chem Phys 15:8952-61|
|Zhong, Yueyang; Feng, Jun; Ruotolo, Brandon T (2013) Robotically assisted titration coupled to ion mobility-mass spectrometry reveals the interface structures and analysis parameters critical for multiprotein topology mapping. Anal Chem 85:11360-8|
|Hyung, Suk-Joon; DeToma, Alaina S; Brender, Jeffrey R et al. (2013) Insights into antiamyloidogenic properties of the green tea extract (-)-epigallocatechin-3-gallate toward metal-associated amyloid-Î² species. Proc Natl Acad Sci U S A 110:3743-8|
|Han, Linjie; Ruotolo, Brandon T (2013) Traveling-wave Ion Mobility-Mass Spectrometry Reveals Additional Mechanistic Details in the Stabilization of Protein Complex Ions through Tuned Salt Additives. Int J Ion Mobil Spectrom 16:41-50|
|Han, Linjie; Ruotolo, Brandon T (2013) Hofmeister salts recover a misfolded multiprotein complex for subsequent structural measurements in the gas phase. Angew Chem Int Ed Engl 52:8329-32|
|Han, Linjie; Hyung, Suk-Joon; Ruotolo, Brandon T (2013) Dramatically stabilizing multiprotein complex structure in the absence of bulk water using tuned Hofmeister salts. Faraday Discuss 160:371-88; discussion 389-403|
|He, Xiaoming; Park, Hyun Min; Hyung, Suk-Joon et al. (2012) Exploring the reactivity of flavonoid compounds with metal-associated amyloid-Î² species. Dalton Trans 41:6558-66|
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