Detecting the dynamics of single biomolecules

Abstract

This thesis is concerned with the detection of single biomolecules. Through detection of individual molecules it can be clarified whether a behavior which has been observed in a population is representative for all molecules. For detection confocal fluorescence microscopy was used, in particular Fluorescence Correlation Spectroscopy (FCS). Complexes of a 217 base-pairs DNA and the fluorescent molecule TMR were immobilized at low concentration on glass coverslips. Through an automatic search single complexes were positioned into the open laser excitation volume (1 [my]m). The detected fluctuating fluorescence intensity of the single molecules was interpreted as association / dissociation of TMR to the nucleotide guanosine. Within the limiting survival time of the fluorophores, the fluctuation rates of the DNA-TMR molecules were found to be heterogeneously distributed. From an analysis of non-fluctuating DNA-TMR complexes an exponential distribution of survival time and number of detected photons was found. Enzymatic activity of horseradish peroxidase (HRP) and of ribonuclease T1 (RNase T1) was analyzed by FCS. For RNase T1, enzyme (~ fM conc.) mediated release of surface immobilized DNA was demonstrated. In the case of HRP, the enzymes were immobilized. The analyzed enzymatic cycle involved formation of a fluorescent enzyme-product complex. It was found that the rate with which the enzyme-product complex was formed was widely distributed. An FCS-setup for ultraviolet excitation and emission was built. With UV-excitation the fluorescence of certain nucleotides and aminoacids can be used. In particular biomolecular dynamics in which such fluorophores are involved could be analyzed. The modified nucleotide 2-aminopurine (2-AP) was used as fluorophore. The diffusion and triplet state dynamics of 2-AP were analyzed. The dynamics of voltage gated ion channels in cell membranes were measured. The voltage- sensing segment S4 of the Shaker potassium channel was labeled with TMR. A combination of the patch-clamp technique and FCS was used. In an allopen or all-closed measurement, correlation between ion current through the channels and fluorescence change generated by S4's conformational rearrangement was observed. This indicates that cross-correlation of the same signals during spontaneous millisecond opening and closing should be detectable.