Multisite Fluorescence in Proteins with Multiple Tryptophan Residues

Abstract

Time-resolved fluorescence experiments were carried out on a variety of apomyoglobins with one or two tryptophan (Trp) residues located at invariant positions 7 and 14 in the primary sequence. In all cases, the Trp fluorescence kinetics were resolved adequately into two discrete lifetime domains, and decay-associated spectra (DAS) were obtained for each decay component. The DAS resolved for unfolded proteins were indistinguishable by position of the emission maxima and the spectral shapes. The folded proteins revealed noticeable differences in the DAS, which relate to the diverse local environments around the Trp residues in the individual proteins. Furthermore, the DAS of wild-type protein possessing two Trp residues were simulated well by that of one Trp mutants either in the native, molten globule, or unfolded states. Overall, employing Trp fluorescence and site-directed mutagenesis allowed us to highlight the conformational changes induced by the single amino acid replacement and generate novel structural information on equilibrium folding intermediates. Specifically, it was found that conformational fluctuations in the local cluster around the evolutionarily conserved Trp 14 are very similar in the native and molten globule states of apomyoglobins. This result indicates that residues in the E and B helices contributing to this cluster are most likely involved in the stabilization of the overall architecture of the structured molten globule intermediate.

Application of conventional molecular dynamics simulation in evaluating the stability of apomyoglobin in urea solution

“…Hydrophobic interactions established among non-polar amino acids ensure the stability of globular proteins in solution by shielding the non-polar amino acids in hydrophobic cores, away from the aqueous environment31. In experimental studies, hydration of the hydrophobic core during protein unfolding and hydrophobic collapse during the start of protein folding are frequently monitored using UV fluorescence spectroscopy912323334353637. This spectroscopy technique exploits the intrinsic fluorescence property of proteins to provide sensitive indications of variation in the solvent accessibility of the hydrophobic core caused by changes in tertiary structure32333435.…”

“…In the case of apoMb, information with regard to structural changes during folding and unfolding is acquired by monitoring the fluctuations in the fluorescence emission of Trp7 and Trp14, both of which are components of the hydrophobic core of apoMb which are confined by Helices A, G and H (Fig. 4A)123233343538.…”

Thermal Denaturation Profiles of Tuna Myoglobin

“…[28][29][30] The stability of sperm whale apoMb was increased by replacement of Pro88 in helix F by Ala, as demonstrated by resistance to proteolysis.…”

Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin

“…The intrinsic fluorescence of Mb originates from Trp-7 and Trp-14, and previous studies have proposed that the increase in fluorescence for the I state is due to either the movement of a quenching amino acid side chain away from Trp-7 or an increase in the flexibility of the indole side chain, allowing it to move into a more apolar environment. The fluorescence of the exposed Trp residues is quenched by surrounding solvent in the completely unfolded U state (11,(61)(62)(63)(64).…”