Picosecond fluorescence decay of tryptophans in myoglobin.

Abstract

The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with F6rster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.Tryptophans are useful optical probes of protein structure because of their relatively long wavelength absorption compared with polypeptides and other common amino acid residues. A first step in realizing the full potential of such probes is the development of techniques to distinguish the different tryptophans in a protein by means of their optical responses. We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1-6), 02 (1,(4)(5)(6), and NO (6, 7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.Pulsed laser techniques are needed to study tryptophan fluorescence in heme proteins as a result of very efficient energy transfer to the heme. The low fluorescence quantum yields for tryptophan in heme proteins (8) suggest that the corresponding lifetimes are in the picosecond range. Thus, to obtain direct information on the lifetimes and motional characteristics of such tryptophans it was necessary to devise experiments by which the fluorescence and its polarization could be measured with picosecond accuracy.Sperm whale (SW) Mb is known to contain the two tryptophan residues, Trp-7 and Trp-14 (9). Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) bu...

Functional electric field changes in photoactivated proteins revealed by ultrafast Stark spectroscopy of the Trp residues

“…This points to a long-lived change in the electrostatic environment of Trp 86 , which lends credence to an ''electrostatic conflict'' (15) To the best of our knowledge, tryptophans have been used in studies of photoinduced energy transfer (35) and electron transfer (36) in proteins, processes that are not or only indirectly related to their function, and as a probe of the solvation dynamics of the interfacial water layer on proteins (37). Here, we demonstrate their use as a reporter of the functional electric field changes within the protein in the course of its biological activity.…”

Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding

“…The [47,48]. The fluorescence decay is dictated by the fast identity of some of the helices; however, as a whole, it represents a more flexible and disordered structure than the native energy transfer from the Trp to the heme, which suppresses all other nonradiative pathways of excited state relaxation.…”

Steady‐State and Picosecond‐Time‐Resolved Fluorenscence Studies on the Recombinant Heme Domain of Bacillus megaterium Cytochrome P‐450

“…The tryptophan fluorescence of the heme proteins is dominated by fast energy-transfer-/decay kinetics with lifetime components in the subnanosecond range (Hochstrasser and Negus, 1984). The efficiency of the energy transfer depends on the distance and the relative orientation of the heme and tryptophan residues, The relative orientation of the two chromophores is expressed through a quantity, IC' (see Materials and Methods).…”

“…However, several studies have been reported on multi-tryptophancontaining proteins, such as myoglobin (Hochstrasser and Negus, 1984;Wills et al, 1990), hemoglobin (Szabo et al, 1984), cytochrome-c oxidase (Das and Mazumdar, 1994), cytochrome P-450,,, (Lange et al, 1994), cytochrome P-45OCzl, and bacteriorhodopsin (Albani et al, 1985;Godik et al, 1993). These studies have proved useful in investigating the nature of the environment around the fluorophore and the relative position of the energy-accepting group with respect to the tryptophan residues, and in determining whether these tryptophan residues lie close to the substrate-binding site.…”