PROTEIN DYNAMICS VIA TIME-RESOLVED CIRCULAR DICHROISM

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A new approach to the detection of circular dichroism (CD) is proposed based on a differential thermal lens design. In this experimental arrangement, the differential absorption characteristic of the CD measurement will be obtained via an optical, rather than an electronic differencing procedure. This approach will provide a substantial improvement in CD detection sensitivity over conventional transmission based instruments. More importantly, the ability to detect the CD information in the time domain of a single laser pulse will permit conformational changes associated with transient phenomena to be studied. Time-resolved CD will be used to study the conformational changes associated with biological electron transfer reactions in cytochrome c and cytochrome oxidase as a function of distance and thermodynamic driving force. Electron transfer will be photoinitiated by excitation of a ruthenium label in cytochrome c, and the structural changes associated with electron transfer within cytochrome c, and from cytochrome c to cytochrome oxidase resolved temporally. The time-resolved CD technique will also be used to study the conformational changes occurring in gramicidin analogs during insertion in a bilayer. Site directed mutagenesis will be used to produce gramicidin derivatives designed with an altered ability to form bilayer channels. Time-resolved CD measurements of these gramicidin derivatives during interaction with the bilayer will address the question as to whether structural or kinetic differences among the analogs can account for the different efficiencies with which they form channels.