Anisotropy decays of protein fluorescence

There is considerable interest in measuring the rotational diffusion and the dynamic properties of proteins. The rates of rotation diffusion can reveal the size and shape of the protein. Also, proteins are known to imdergo structural fluctuations, a topic which has been broadly studied by both experimentation and computer simulations [32-36], The time-resolved experiments are often directed towards a comparison of the measurable dynamics of proteins with the calculated dynamics. One promising approach is to use anisotropy data from intrinsic protein fluorescence. If such data are available with picosecond resolution then such a comparison should be possible. 

In the time-domain the anisotropy decay is obtained from the time-resolved decays of the parallel and perpendicular polarized components of the emission. More specifically, one measures the time-resolved decays of the parallel ( ) and the perpendicular ( ) components of the emission, and calculates the time-resolved anisotropy. 

In this expression r(/) is the time-dependent anisotropy, 0 the correlation times and g, the fraction of the total anisotropy (r ) which decays with this correlation time. In general we expect one component (tf,) due to rotational diffusion of the protein, and one due to torsional motions of the tryptophan residue, if such motions are significant. In proteins which contain more than a single fluorescent residue there can be energy transfer among the residues, which can appear as a component in the anisotropy decay. The timescale of energy transfer depends upon the distance and orientation between the residues, but there is little information on the timescale of energy transfer between intrinsic fluorophores in proteins. 

The measurements are different in the frequency-domain. In this case we measure the phase shift between the parallel and perpendicular components of the emission, and a frequency-dependent anisotropy, which is analogous to the steady state anisotropy. These two types of data are used to determine the decay law for the anisotropy (equation 15). 

Mehttin and Nuclease illustrate how the anisotropy decay is reflected in the frequency-domain data. From earlier studies it was known that the single tryptophan residue in Sj Nuclease was mostly rigid [36], so that its anisotropy decay should display a single correlation time for rotational diffusion near 11 nsec. In contrast, melittin monomer is thought to be disordered in aqueous solution, so that a rapid anisotropy decay is expected due to local tryptophan motions. 

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J. R. Lakowicz, G. Laszko, I. Gryczynski, and H. Cherek, Measurement of subnanosecond anisotropy decays of protein fluorescence using frequency-domain fluorometry, J. Biol. Chem. 261, 2240-2245 (1986). 

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