Identification of Subunit-Subunit Interaction Sites in αA-WT Crystallin and Mutant αA-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry

We determined the interaction sites of the cytochrome P450's protein-partners: 2B4 (d-2B4) and NADPH-cytochrome P450 of reductase (d-Fp). While in operation, these proteins are forming the complexes. We used 4-4'-dithio(bisphenyl)azide linker for non-specific covalent coupling of d-2B4 complexes with d-Fp in Emulgen-913 - monomerized system. Covalently-linked peptides in this complex were identified with ESI-MS/MS. Several sites of these proteins' binding with each other were revealed. Based on them, a model of intermolecular protein interactions was created. The model includes 5 cross-linker-stabilized contact sites of d-2B4 with d-Fp involving the following peptides of d-2B4 and d-Fp: 1) d-2B4423-433 и d-Fp 102-109; 2) d-2B4324-336 и d-Fp570-585; 3) d-2B4327-336 и d-Fp452-464; 4) d-2B4 192-197 и d-Fp456-464; 5) d-2B4 134-139 и d-Fp406-425.Herein, in the latter two cases, the peptides of d-Fp are located in their inter-domain slit and stabilize protein-protein complex via nanoprobe cross-linker; therefore, the formation of d-2B4/d-Fp complexes in these sites may involve aminoacid residues d-Fp456-464 and d-Fp406-425 surrounding inter-domain slit.

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Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking

Analytical Chemistry ◽

10.1021/acs.analchem.6b02886 ◽

2016 ◽

Vol 88 (20) ◽

pp. 10215-10222 ◽

Cited By ~ 18

Author(s):

Jayanta K. Chakrabarty ◽

Aishwarya G. Naik ◽

Michael B. Fessler ◽

Gerhard R. Munske ◽

Saiful M. Chowdhury

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Cross Linking ◽

Tandem Mass ◽

High Confidence ◽

New Approach ◽

Cross Linker ◽

Protein Cross Linking

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E. coli GyrA Tower Domain Interacts with QnrB1 Loop B and Plays an Important Role in QnrB1 Protection from Quinolone Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00402-21 ◽

2021 ◽

Author(s):

Chunhui Chen ◽

Yin Wang ◽

Hidemasa Nakaminami ◽

Eu Suk Kim ◽

George A. Jacoby ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acids ◽

Amino Acid ◽

Dna Gyrase ◽

Amino Acid Residues ◽

Site Specific ◽

E Coli ◽

Small Deletion ◽

Interaction Sites ◽

Repeat Proteins

The Qnr pentapeptide repeat proteins interact with DNA gyrase and protect it from quinolone inhibition. The two external loops, particularly the larger loop B, of Qnr proteins are essential for quinolone protection of DNA gyrase. The specific QnrB1 interaction sites on DNA gyrase are not known. In this study, we investigated the interaction between GyrA and QnrB1 using site-specific photo crosslinking of QnrB1 loop B combined with mass spectrometry. We found that amino acid residues 286-298 on the Tower domain of GyrA interact with QnrB1 and play a key role in QnrB1 protection of gyrase from quinolone inhibition. Alanine replacement of arginine at residue 293 and a small deletion of amino acids 286-289 of GyrA resulted in a decrease in the QnrB1-mediated increase in quinolone MICs and also abolished the QnrB1 protection of purified DNA gyrase from ciprofloxacin inhibition.

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A Fast Lysine Cross-linker DOPA Enables Mass Spectrometry Analyses of Protein Unfolding and Weak Protein-protein Interactions

10.1101/2020.11.05.369280 ◽

2020 ◽

Author(s):

Jian-Hua Wang ◽

Yu-Liang Tang ◽

Rohit Jain ◽

Fan Xiao ◽

Zhou Gong ◽

...

Keyword(s):

Mass Spectrometry ◽

Conformational Changes ◽

Protein Interactions ◽

Guanidine Hydrochloride ◽

Rnase A ◽

Mass Spectrometry Analysis ◽

Cross Linking ◽

Protein Protein Interactions ◽

Protein Structure Analysis ◽

Cross Linker

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.

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Photoreactive Stapled Peptides to Identify and Characterize BCL-2 Family Interaction Sites by Mass Spectrometry

Regulated Cell Death Part A: Apoptotic Mechanisms - Methods in Enzymology ◽

10.1016/b978-0-12-417158-9.00002-9 ◽

2014 ◽

pp. 25-48 ◽

Cited By ~ 6

Author(s):

Susan Lee ◽

Craig R. Braun ◽

Gregory H. Bird ◽

Loren D. Walensky

Keyword(s):

Mass Spectrometry ◽

Family Interaction ◽

Stapled Peptides ◽

Interaction Sites

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Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011630 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7595-7607 ◽

Cited By ~ 1

Author(s):

Laura N. Jeffreys ◽

Kamila J. Pacholarz ◽

Linus O. Johannissen ◽

Hazel M. Girvan ◽

Perdita E. Barran ◽

...

Keyword(s):

Mass Spectrometry ◽

Cytochrome P450 ◽

Bound States ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Full Length ◽

P450 Bm3 ◽

Interaction Sites ◽

Domain Interactions ◽

Hdx Ms

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

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