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 reporter of the conformational change. Furthermore, we found that the time-dependent increase in mass, owing to free-radical oxidation, is a measure of the rate constant for the transition from the unfolded to the first intermediate state. This advance offers the promise that, when extended with mass-spectrometry based proteomic analysis, the sites and kinetics of folding/unfolding can also be followed at sub millisecond times.Deciphering protein folding is essential for understanding biological processes and developing therapeutic approaches to misfolding-related diseases 1-3 . Protein folding can be followed in equilibrium experiments, which monitor protein states as a function of temperature or denaturant concentration, and in kinetics, to give a time-dependent conformational change.Starting in the early 1990s, mass spectrometry (MS) emerged as an effective tool for supporting both thermodynamic and kinetic protein-folding studies. The advantage of modern MS is its ability to measure extents of protein modification and pinpoint their locations. For pulsed H/D amide exchange and other pulsed covalent labeling kinetics 4, 5 , MS detection can now track folding events down to the millisecond timescale. Protein folding, however, often occurs more rapidly (in microseconds or even less 6 ), which has been a difficult range to access in current MS-based studies.Here we describe a new approach to investigate protein folding. We use two lasers, one to provide a temperature-jump (T-jump) and a second to generate reagents to footprint the consequences of the T-jump. This is an example of "pump/probe" 7, 8 , but it is distinguished by the use of chemical reactions as the structural probe rather than the usual spectroscopic approaches. To our knowledge, the first irreversible labeling accompanying protein conformational change was demonstrated by Jha and Udgaonkar 9 , who explored protein folding on the msec timescale by combining mutagenesis, pulsed thio-labeling and global protein analysis by MS. Recently, Konermann et al. combined rapid mixing and fast photochemical oxidation of proteins (FPOP) to observe protein unfolding 10 /folding 11 intermediates at times in the 10 ms range. Our approach takes advantage of FPOP, developed in our laboratory 12 , whereby pulsed laser-photolysis yields hydroxyl radicals that mgross@wustl.edu. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 November 10. We chose barstar (10.3 kDa) as a test protein because it is denatured at 0 °C and fo...

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…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).…”

“…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.…”

“…Secondly, FPOP holds promise for examining short-lived protein folding intermediates [33]. FPOP has been adopted by various researchers, and it has been applied to a range of protein systems [34][35][36][37][38][39][40][41][42][43][44][45].…”

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

“…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).…”

Molecular dynamics simulation exploration of unfolding and refolding of a ten-amino acid miniprotein

“…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).…”