Elucidation of the mechanisms by which proteins fold to attain their native structure remains one of the most challenging problems in biochemistry. Recently, many diseases have been linked to possible protein misfolding followed by a loss/alteration of its function and/or aggregation (amyloidosis). There is also a very close relationship between the folding/conformational stability of proteins and their ligand- binding properties. We are seeking to further our understanding of the dynamics of the folding process, as well as conformational stability of proteins and protein-ligand complexes. To that end, we will use electrospray (ESI) mass spectrometry (MS) to study folding processes in vitro. We will employ hydrogen/deuterium (H/D) exchange in solution to probe the conformational stability and folding/unfolding dynamics of proteins under various conditions. Transiently populated intermediate states will be detected in these experiments and characterized by different degrees of backbone amide protection. Because of the high data acquisition rate, mass spectrometry offers a facile way to resolve these intermediates on a time scale from tens of msec to hours. The global information on the protein conformational stability and folding dynamics will be complemented by local (residue-specific) information. Several approaches will be used to achieve that goal. In the first approach, collisionally activated dissociation (CAD) of protein ions will be used to measure the deuterium content locally as a function of exchange time. Particular care will be taken to ensure that ion-molecular processes in the gas phase (e.g., hydrogen scrambling and/or H/D exchange with residual solvent molecules) do not influence the measurements. This approach will be particularly useful for studying the folding events on a time scale of tens of seconds to hours, as it allows real time monitoring to be carried out. In another approach, the HID exchange will be quenched at a certain time (by lowering the solution pH and temperature) and extent of the H/D exchange will be analyzed by using ESI/CAD or peptic digest followed by ESI MS analysis. This will allow us to extend our studies to a sub-second time scale. We will also use chemical cross-linking to elucidate the topology of ligand binding sites in proteins, as well as inter-domain interactions in multi-domain proteins. These methods will be applied to study folding dynamics and conformational stability of several proteins. Molecular mechanisms of ligand binding to Cellular Retinoic Acid Binding Protein I (CRABP I) will be studied by mapping the energy landscape for both apo- and holo-forms of the protein. We will also study mechanisms of ferric ion binding/release by iron transporting proteins from the transferrin family. The proposed studies will significantly further our understanding of the protein folding processes and the mechanisms by which transport proteins function in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061666-05
Application #
6769339
Study Section
Special Emphasis Panel (ZRG1-BMT (01))
Program Officer
Edmonds, Charles G
Project Start
2000-07-01
Project End
2005-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
5
Fiscal Year
2004
Total Cost
$191,875
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Zhao, Hanwei; Wang, Shunhai; Nguyen, Son N et al. (2016) Evaluation of Nonferrous Metals as Potential In Vivo Tracers of Transferrin-Based Therapeutics. J Am Soc Mass Spectrom 27:211-9
Fatunmbi, Ololade; Abzalimov, Rinat R; Savinov, Sergey N et al. (2016) Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry. Biochemistry 55:1918-28
Pawlowski, Jake W; Kellicker, Noelle; Bobst, Cedric E et al. (2016) Assessing the iron delivery efficacy of transferrin in clinical samples by native electrospray ionization mass spectrometry. Analyst 141:853-61
Wang, Shunhai; Bobst, Cedric E; Kaltashov, Igor A (2015) A new liquid chromatography-mass spectrometry-based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous systems. Eur J Mass Spectrom (Chichester) 21:369-76
Wang, Shunhai; Kaltashov, Igor A (2015) Identification of reduction-susceptible disulfide bonds in transferrin by differential alkylation using O(16)/O(18) labeled iodoacetic acid. J Am Soc Mass Spectrom 26:800-7
Wang, Guanbo; Kaltashov, Igor A (2014) Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry. Anal Chem 86:7293-8
Bobst, Cedric E; Kaltashov, Igor A (2014) Enhancing the quality of H/D exchange measurements with mass spectrometry detection in disulfide-rich proteins using electron capture dissociation. Anal Chem 86:5225-31
Kaltashov, Igor A; Bobst, Cedric E; Nguyen, Son N et al. (2013) Emerging mass spectrometry-based approaches to probe protein-receptor interactions: focus on overcoming physiological barriers. Adv Drug Deliv Rev 65:1020-30
Nguyen, Son N; Bobst, Cedric E; Kaltashov, Igor A (2013) Mass spectrometry-guided optimization and characterization of a biologically active transferrin-lysozyme model drug conjugate. Mol Pharm 10:1998-2007
Wang, Guanbo; Abzalimov, Rinat R; Bobst, Cedric E et al. (2013) Conformer-specific characterization of nonnative protein states using hydrogen exchange and top-down mass spectrometry. Proc Natl Acad Sci U S A 110:20087-92

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