Our goal is to produce a commercial instrument which combines the laser- induced photolysis of photolabile caged compounds and acoustic detection to obtain very fast kinetic information on biological molecules. In Phase I we built a prototype pulsed-laser photoacoustic instrument and showed that it could be used to measure the rates associated with the binding of protons to polypeptides. We now propose instrumental and software improvements that will dramatically extend the time range over which we can make measurements, as well as the quality of information that we can obtain. Using the improved instrumentation, we will perform a survey of the kinetics of release for commercially available caged compounds, extending from the nitrophenylethyl(NPE-) caged proton we used in our Phase I studies, to the numerous faster releasing caged compounds currently available. We will also continue and extend our polypeptide studies, adding control experiments and obtaining corroborating evidence of molecular relaxations using other kinetic approaches. The product of our work will provide researchers with a new tool to measure molecular relaxations by their kinetics, enthalpic changes, and volumetric changes, in a very important submillisecond time range, without the requirements of an intrinsic molecular chromophore.
The proposed instrument will be used to measure very fast kinetics of biological molecules on submillisecond time scales. Thus, it can serve the research community as a very fast alternative to stopped flow. As a general purpose photoacoustic device, it has many additional applications in a wide range of fields including pharmaceutical, photochemical, photophysical and environmental research.
| Oertling, W Anthony; Cornellison, Charisa D; Treff, Nathan R et al. (2007) Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase. J Inorg Biochem 101:635-43 |
| Abbruzzetti, S; Viappiani, C; Small, J R et al. (2001) Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique. J Am Chem Soc 123:6649-53 |
| Abbruzzetti, S; Crema, E; Masino, L et al. (2000) Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump. Biophys J 78:405-15 |
| Abbruzzetti, S; Viappiani, C; Small, J R et al. (2000) Kinetics of local helix formation in poly-L-glutamic acid studied by time-resolved photoacoustics: neutralization reactions of carboxylates in aqueous solutions and their relevance to the problem of protein folding. Biophys J 79:2714-21 |
