2010
DOI: 10.1021/ja106518d
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Temperature Jump and Fast Photochemical Oxidation Probe Submillisecond Protein Folding

Abstract: We report a new mass spectrometry-based approach to study protein-folding dynamics at the sub millisecond time. The strategy couples temperature jump with fast photochemical oxidation of proteins (FPOP) whereby folding/unfolding is followed by changes in oxidative modifications by OH radical reactions. Using a flow system containing the protein barstar as a model, we altered the protein's equilibrium conformation by temperature jump and demonstrated that its reactivity with OH free radicals serves as a reporte… Show more

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Cited by 78 publications
(118 citation statements)
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References 17 publications
(32 reference statements)
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“…FPOP of acid-unfolded apo-Mb produces F U = 4.3%, much lower than the expected value of 25% (Figure 5b). Similarly, FPOP of semi-unfolded barstar with EVF = 25% [63] resulted in F U = 7% (Figure 2a in reference [41]). It is concluded that F U values in FPOP tend to be too low (i.e., the percentage of oxidized protein is higher than expected).…”
Section: Fpop Mass Spectramentioning
confidence: 90%
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“…FPOP of acid-unfolded apo-Mb produces F U = 4.3%, much lower than the expected value of 25% (Figure 5b). Similarly, FPOP of semi-unfolded barstar with EVF = 25% [63] resulted in F U = 7% (Figure 2a in reference [41]). It is concluded that F U values in FPOP tend to be too low (i.e., the percentage of oxidized protein is higher than expected).…”
Section: Fpop Mass Spectramentioning
confidence: 90%
“…Perhaps this is because earlier FPOP work largely focused on native proteins where many side chains are protected by burial, such that overoxidation artifacts are difficult to recognize. Unfolded proteins (as in Figure 5b and reference [41]) are more conducive to stringent F U versus EVF tests because of their greatly elevated reactivity.…”
Section: Fpop Mass Spectramentioning
confidence: 99%
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“…42 It should be noted that ultra-fast HRF techniques, such as those using nanosecond photolysis of peroxide (FPOP), may overcome some of these limitations as well. 61,62 Homology modeling A molecular model of the mAb was generated using homology modeling, allowing us to explore the relationship between reactivity of the individual probes against their predicted solvent accessibility profile. Figure 4 shows the homology model of the protein constructed using Swiss-Model workspace based on available templates of intact hetero-tetrameric IgG1 mAb (PDB ID:1I GY) and Fab fragment (PDB ID:1BJ1).…”
Section: Linearity Under High Dose Conditionsmentioning
confidence: 99%
“…Hydrogen-exchange mass-spectrometry can track the formation of secondary structures during folding by measuring how easily protons (H ? ions) are exchanged between water and amino acids in the proteins (Tsui et al 1999;Eyles and Kaltashov 2004;Pan et al 2010;Chen et al 2010;Gruebele 2010). However, a detailed understanding of folding is still missing due to the high dimensionality of the protein conformational spaces and by the wide range of relevant time scales (Daniel et al 2003).…”
Section: Introductionmentioning
confidence: 99%