Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence

Abstract

AHSTRAC r: The fluorescence decay of chicken pepsinogen is not monoexponential throughout the emission spectrum. For light emitted a t the long wavelength region of the fluorescence spectrum, the decay can be described by two exponential terms, one of them exhibiting a negative amplitude. l'his behavior shows that in this region of the spectrum the fluorescence builds up before it decays, indicating that the electronically excited species involved has been created during the fluorescence lifetime. For a variety of reasons it seems very unlikely that this emitting species as formed by energy transfer from other entities in the protein. The buildup of the fluorescence at the red edge of the spectrum prior to emission thus reflects a genuine relaxation process i n the protein molecule in the nanosecond time scale. the exc,i:ed chromophore shifting its emission spectrum to the red in the course of the relaxation process. The reaction in-T f i e fluorescence emitted by molecules in condensed phases is invariably shifted to longer wavelengths relative to the absorption spectrum. The larger the interaction between the excited chroniophore with its environment the greater the shift of the fluorescence band to the red. This behavior of fluorescence spectra is due to relaxation processes which the excited molecule undergoes prior to the emission process, the rate of relaxation being usuallj. much faster than the rate of light emission. The processes which take place in the excited state may be of a variety of kinds: dissipation of vibrational energy or change of conformation of the electronicall4 excited molecules, interactions with surrounding solvent molecules, association or dissociation reactions in the excited state, or processes of fast energy transfer from one chrornophoric group to another in the system studied. Part of the energy of the excited molecules is lost in these processes and the photon emitted subsequently is left N i t h less energ) t o carry; the emitted light is thus of longer wavelerig t h .I n all cases in which the reactions in the excited state are much faster than the rate of light emission, the fluorescence collected at the various wavelengths of the spectrum deca) s ni ono t on I) us1 y with ti iri e. IJ nder certain circumstances son1 e of t h e relaxation processes are not fast compared t o the lifetime of the excited state. Such a situation is disclosed by buildup of the fluorescence intensity prior to the decay at some region of the emission spectrum. Examples of this kind were reported when the electronically excited molecule undergoes association reactions (Speed and Selinger. 1969) volved in the relaxation may be a nonspecific orientation of various groups around the excited chromophore or a forniation of a more specific excited state complex, i.e.. an exciplex. This nanosecond relaxation process is Conformation dependent and disappears upon denaturation. Similarly, chicken pepsin a t neutral pH fails to show it. This sce1m to be the first case in which a relaxation process in the iianos...

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins

“…The heterogeneity of the emission spectra arises either from at least two ground-state conformers with different excited-state behaviour or from the presence of an excited-state reaction on the nanosecond timescale. This reaction is not yet defined but it can originate from exciplex formation (Grinvald and Steinberg 1974), solvent relaxation (Gudgin-Templeton and Ware 1984) or in general, from relaxation into a different protein environment. A generalized scheme involving a uni-directional excited-state reaction and possibly consistent with the experimental data, for D. gigas at least, is given in Fig.…”

Thermodynamic fluctuations in protein molecules.

“…Experimental techniques such as fluorescence quenching (1,2) and relaxation (3), phosphorescence (4), nuclear magnetic resonance (5-7) point to a rather fluid, dynamic structure for globular proteins involving rapid conformational fluctuations which allow relatively easy, if somewhat transient, accessibility of interior groups to solvent and molecular probes (1). Some aspects of the dynamics of protein molecules have been recently reviewed (8).…”

“…[3] A representative globular protein of molecular weight 25,000 has a mass of 4 The compressibility ((3T) of proteins in solution is not known, but is probably less than 5 X 10-6 atm-1 (0.5 Pa) (22) (i.e., much less than (3T for organic liquids, and approaching that of solids).…”

Dynamics of a protein matrix revealed by fluorescence quenching.

“…Evidence for some type of motion occurring at a time interval as small as a fraction of a nanosecond has been obtained from nuclear magnetic resonance (1), nitroxide spin labeling (2), dielectric relaxation (3), and various fluorescence studies (4)(5)(6). Depending on the time domain and experimental approach, descriptive terms such as "breathing" (7), "relaxation," "segmental motion," and "mobile defect" (8) have been advanced to portray the conformational "motility" (9) of proteins.…”

“…Fluorescence measurements were made with a Farrand Mark I spectrofluorometer using 5 (16). [1] In this equation, F0/F is the ratio of the fluorescence yield (or intensity) in the absence and presence of a given concentration of quencher [Q], and K is the quenching constant.…”