A picosecond IR laser (PIRL) can be used to blast proteins out of tissues through desorption by impulsive excitation (DIVE) of intramolecular vibrational states of water molecules in the cell in less than a millisecond. With PIRL-DIVE proteins covering a range of a few kDa up to several MDa are extracted in high quantities compared to conventional approaches. The chemical composition of extracted proteins remains unaltered and even enzymatic activities are maintained.

Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5′-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10−7 − 10−1 s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5′-deoxyadenosyl radical pair quantum yields of <0.01 at 10−6 s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, Pf, Ps, and Pc. The relative amplitudes and first-order recombination rate constants of Pf (0.4-0.6; 40-50 s−1), Ps, (0.3-0.4; 4 s−1) and Pc (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not “switch” the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5′-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.

The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl) in coenzyme B 12 (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10 -7 -10 -1 s time scale at 295 K by using transient ultraviolet-visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin-5′-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt-carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10 -7 s for cob(II)alamin of 0.08 ±0.01, which is 3-fold less than for AdoCbl in aqueous solution (0.23 ±0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, P f , P s , and P c , are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: P f (2.2×10 3 s -1 ; 0.02), P s (4.2×10 2 s -1 ; 0.01), and P c (constant amplitude, no recombination; 0.05). Binding of the substrate analog, (S)-1-amino-2-propanol, to EAL eliminates the P f state, and lowers the quantum yield of P c (0.03) relative to P s (0.01), but does not significantly change the quantum yield or decay rate constant of P s , relative to holo-EAL. The substrate analog thus influences the quantum yield at 10 -7 s by changing the cage escape rate from the geminate cob(II)alamin-5′-deoxyadenosyl radical pair state. However, the predicted substrate analog binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analog does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.Coenzyme B 12 -dependent enzymes catalyze radical mediated rearrangement reactions in both bacteria and mammals (1-3). The first step in the catalytic cycle is the homolytic cleavage of the cobalt-carbon (Co-C) bond in coenzyme B 12 (adenosylcobalamin, AdoCbl; Figure 1), which results in the formation of the cob(II)alamin-5′-deoxyadenosyl radical pair. The C5′ radical center of the 5′-deoxyadenosyl moiety then migrates over 5-6 Å (4,5) to abstract a † The project described was supported by Grant Number R01DK054514 from the National Institute of Diabetes and Digestive and Kidney Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health. * Corresponding Author: Kurt Warncke Department of Physics N201 Mathematics and Science Center 400 Dowman Drive Emory University Atlanta, Georgia 30322-2430 kwarncke@physics.emory.edu Phone: 404-727-2975 Fax: 404-727-0873 SUPPORTING INFORMATION AVAILABLE Monoexponential plus consta...

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation.Biological significanceEnzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen.