Red-Edge Excitation Shifts

Chattopadhyay A, Mukherjee S (1999) Red edge excitation shift of a deeply embedded membrane probe implications in water penetration in the bilayer. J Phys Chem B 103(38) 8180-8185... 

Haidar S, Chattopadhyay A (2007) Dipolar relaxation within the protein matrix of the green fluorescent protein a red-edge excitation shift study. J Phys Chem Bill 14436-9... 

The red-edge excitation spectra and anisotropy measurements yield information on the dynamics of the fluorophore and of its environment. At high concentration of calcofluor compared to that of a i-acid glycoprotein, a red-edge excitation shift is observed (Fig. 8.23a). This shift indicates that the microenvironment of the fluorophore exhibits restricted motions. Therefore, the carbohydrate residues in the vicinity of calcofluor are rigid and do not show any segmental motions. 

Chattopadhyay A, Mukherjee S (1993) Fluorophore environments in membrane-bound probes— red edge excitation shift study. Biochemistry 32(14) 3804-3811. doi 10.1021/ bi00065a037... 

Observation of reorientational dynamics of dipolar groups surrounding the fluorophore in response to changes in the dipole moment of the fluorophore occurring upon electronic excitation. Such dynamics result in the appearance of spectral shifts with time,(1 ) in changes of fluorescence lifetime across the fluorescence spectrum,(7,32) and in a decrease in the observable effects of selective red-edge excitation.(1,24 33 34) The studies of these processes yield a very important parameter which characterizes dynamics in proteins— the reorientational dipolar relaxation time, xR. 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

These data may be explained in terms of the above mechanism of the long-wavelength shift of fluorescence spectra for red-edge excitation. The properties of the environment of the tryptophan residues in the proteins studied are such that during the lifetime of the excited state, structural relaxation of the surrounding dipoles fails to proceed. Studies of the dependence of the... 

Red-edge excitation spectra method is very sensitive to the changes that occur in the microenvironment of the Trp residues. For example, Trp residues of intact lens protein from rat exhibit an emission shift of 14 nm upon varying the excitation from 290 to 308 nm. Photodamaging the lens induces an emission shift of 24 nm (Rao et al. 1989). 

Rotation of the crystal around the excitation beam by steps of 45 5° does not induce any modification in the red-edge excitation spectra results. At all angles (0, 45, 90, 135, and 180°), no shift was observed either in the fluorescence maximum or in the center of gravity of the spectrum (data not shown). This result indicates clearly that at all the angles we are monitoring the dynamics of the same microenvironments of the Trp residues in the crystal of protein. At all angles, we are exciting the dipoles of the two buried Trp residues. 

Growth of the degree of fluorescence polarization (the Weber s effect) and a decrease of energy transfer efficiency while shifting the excitation wavelength to the red edge. 

It should be recalled that, in polar rigid media, excitation on the red-edge of the absorption spectrum causes a red-shift of the fluorescence spectrum with respect to that observed on excitation in the bulk of the absorption spectrum (see the explanation of the red-edge effect in Section 3.5.1). Such a red-shift is still observable if the solvent relaxation competes with the fluorescence decay, but it disappears in fluid solutions because of dynamic equilibrium among the various solvation sites. 

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