Hypercoiled conformation

Time-resolved fluorescence spectroscopy and fluorescence anisotropy measurements have been applied to study (i) excimer formation and energy transfer in solutions of poly(acenaphthalene) (PACE) and poly(2-naphthyl methacrylate) (P2NMA) and (ii) the conformational dynamics of poly(methacrylic acid) (PMA) and poly (acrylic acid) as a function of solution pH. For PACE and P2NMA, analysis of projections in which the spectral, temporal and intensity information are simultaneously displayed have been used to re-examine kinetic models proposed to account for the complex fluorescence decay behaviour that is observed. Time-resolved fluorescence anisotropy measuranents of fluorescent probes incorporated in PMA have led to the proposal of a "connected cluster" model for the hypercoiled conformation of this polymer existing at low pH. 

A further application of time-resolved fluorescence measurements is in the study of conformational dynamics of polymer chains in solution. Fluorescence anisotropy measurements of macromolecules incorporating suitable fluorescent probes can give details of chain mobility and polymer conformation (2,14). A particular example studied in this laboratory is the conformational changes which occur in aqueous solutions of polyelectrolytes as the solution pH is varied (15,16). Poly(methacrylic acid) (PMA) is known to exist in a compact hypercoiled conformation at low pH but undergoes a transition to a more extended conformation at a degree of neutralization (a) of 0.2 to 0.3 (1 6). Similar conformational transitions are known to occur in biopolymer systems and consequently there is considerable interest in understanding the nature of the structures present in model synthetic polyelectrolyte solutions. 

Comparison of the transient fluorescences at pH 5.7 (Fig. 2.2) and pH 3.2 (Fig. 2.3) reveals that the duration of the decay has increased at low degrees of ionization. This is consistent with solubilization of pyrene in the protective domains of the hypercoiled conformation. 

Examination of the kq values listed in Table 2.2 clearly reveals the conformational transition of the poly electrolyte for example, PMAA has been labeled with 1-pyrene acrylic acid (PyAA) [22], ACE [12], 1VN [12], and vinyl diphenylanthracene (VyDPA) [94] and quenched with nitromethane in aqueous media. The kq values derived from lifetime data for each of these labels are of the order of 0.5 x 109 moE1 dm3 s 1 at low degrees of ionization. The formation of the hypercoiled conformation provides a degree of protection for the excited state from the quencher, and a low value of kq results. 

When TRAMs are made on dispersed probes, tc will reflect the speed of rotation of the fluorophore [20,46,60,76], which can be related to the microviscosity of the medium. In the context of probing the structure of PMAA, the fluorescent dyes have been occluded in the hypercoiled conformation [46,60,76] allowing an estimate of the size of the rotating, solubilizing cluster to be derived from the resultant tc. Alternatively, if the fluorophore is covalently attached to the polyelectrolyte in the form of a label then, depending on its mode of attachment, information concerning motion of the chain ends [46,60,76], the backbone [26,88,112,113], and chain substituents [26,88] can be derived from tc. 

For the probes 9-methyl anthracene (MA) and DMA solubilized in the hypercoiled conformation of PMAA, the anisotropy decays could best be described by invoking double-exponential analysis of the form described by Equation 2.32... 

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