Laser flash photochemical oxidation to locate heme binding and conformational changes in myoglobin

Hambly, David M.; Gross, Michael L.

doi:10.1016/j.ijms.2006.08.018

Cited by 60 publications

(114 citation statements)

References 59 publications

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“…FPOP used an excimer laser to photolyze hydrogen peroxide to give two OH radicals; the H 2 O 2 was added in low concentration to the protein solution, to form hydroxyl radicals [44, 46]. To control the radical lifetime, a pulsed laser and selected scavenger of 20 mM Gln were used for protein labeling at a microsecond time scale, faster than protein unfolding [45].…”

Section: Methodsmentioning

confidence: 99%

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Yan

Chen

Wei

et al. 2014

J. Am. Soc. Mass Spectrom.

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Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the tenth type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human EGFR, we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFRAddnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitope from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

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Section: Methodsmentioning

confidence: 99%

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Yan

Chen

Wei

et al. 2014

J. Am. Soc. Mass Spectrom.

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“…The Δ40APE1sample was diluted with water to a final concentration of 1 μm and then digested using a protein:trypsin ratio of 50:1 at 37 °C for 4 h. Native digestion conditions were similar to those successfully used for other problems involving purified protein samples(23, 24). The solution was then analyzed by LC-MS/MS whereby 5 μL of digestion solution was consumed for each experiment.…”

Section: Methodsmentioning

confidence: 99%

Interactions of Apurinic/Apyrimidinic Endonuclease with a Redox Inhibitor: Evidence for an Alternate Conformation of the Enzyme

Delaplane²,

Luo

et al. 2010

Biochemistry

108

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Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction/oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried Cys residues 65 and 93 are critical for APE1’s redox activity. To further detail the redox mechanism, we developed a chemical footprinting/mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When incubated with E3330, two NEM-modified products were observed, one with 2 and a second with 7 added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen-deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase disulfide bond formation involving redox critical Cys residues in APE1 as assessed by LC-MS/MS, suggesting a basis for its inhibitory effects on APE1’s redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme.

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“…We report here a method that combines carboxyl group modification with mass spectrometry to afford surface mapping or footprinting (29,30) of the protein, revealing the interaction of proteins associated with membranes. We chose the reagent, glycine…”

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confidence: 99%

Membrane orientation of the FMO antenna protein from Chlorobaculum tepidum as determined by mass spectrometry-based footprinting

Wen¹,

Zhang

Gross

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

194

228

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The high excitation energy-transfer efficiency demanded in photosynthetic organisms relies on the optimal pigment-protein binding orientation in the individual protein complexes and also on the overall architecture of the photosystem. In green sulfur bacteria, the membrane-attached Fenna-Matthews-Olson (FMO) antenna protein functions as a ''wire'' to connect the large peripheral chlorosome antenna complex with the reaction center (RC), which is embedded in the cytoplasmic membrane (CM). Energy collected by the chlorosome is funneled through the FMO to the RC. Although there has been considerable effort to understand the relationships between structure and function of the individual isolated complexes, the specific architecture for in vivo interactions of the FMO protein, the CM, and the chlorosome, ensuring highly efficient energy transfer, is still not established experimentally. Here, we describe a mass spectrometrybased method that probes solvent-exposed surfaces of the FMO by labeling solvent-exposed aspartic and glutamic acid residues. The locations and extents of labeling of FMO on the native membrane in comparison with it alone and on a chlorosome-depleted membrane reveal the orientation. The large differences in the modification of certain peptides show that the Bchl a #3 side of the FMO trimer interacts with the CM, which is consistent with recent theoretical predictions. Moreover, the results also provide direct experimental evidence to confirm the overall architecture of the photosystem from Chlorobaculum tepidum (C. tepidum) and give information on the packing of the FMO protein in its native environment.chemical labeling ͉ energy transfer ͉ FMO protein ͉ mass spectrometry ͉ protein footprinting P hotosynthesis is a fundamental biological process that harvests solar energy to power life on Earth (1). A diverse family of pigment-protein complexes and elegant architectures accomplish the necessary light-harvesting and energy-storage processes (2-5). In photosynthetic green sulfur bacteria, light absorbed by a large antenna complex known as a chlorosome (6-8) is transferred through a protein called Fenna-Matthews-Olson (FMO) (9) to the reaction center, which is embedded in the cytoplasmic membrane (CM). Together, they form a funnel-like architecture to facilitate energy transfer. The specific orientation of the critical linker, the FMO protein, however is unknown (Fig. 1A).The structure of the FMO protein was the first (bacterio)chlorophyll binding protein to be determined by X-ray crystallography. Structures of this protein from 2 species, Prosthecochloris aestuarii 2K (10, 11) and Chlorobaculum tepidum (12) are now available, and they show strong structural and spectral similarities. The FMO protein consists of 3 identical subunits of mass 40 kDa related by a 3-fold axis of symmetry. The 3 monomers form a disc with a C3 symmetry axis perpendicular to the disc plane (Fig. 1B). There are 7 BChl a molecules in a monomer, although an eighth pigment has been resolved in newly solved structures (13,14). Eac...

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Laser flash photochemical oxidation to locate heme binding and conformational changes in myoglobin

Cited by 60 publications

References 59 publications

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

Interactions of Apurinic/Apyrimidinic Endonuclease with a Redox Inhibitor: Evidence for an Alternate Conformation of the Enzyme

Membrane orientation of the FMO antenna protein from Chlorobaculum tepidum as determined by mass spectrometry-based footprinting

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