Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human beta-cardiac myosin
JOURNAL OF EXPERIMENTAL BIOLOGY
2016; 219 (2): 161-167
Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State
2013; 105 (10): 2392-2402
Reduced Fluorescence Lifetime Heterogeneity of 5-Fluorotryptophan in Comparison to Tryptophan in Proteins: Implication for Resonance Energy Transfer Experiments
JOURNAL OF PHYSICAL CHEMISTRY B
2011; 115 (22): 7479-7486
Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N(∗). Fluorescence resonance energy transfer measurements show that N(∗) then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N(∗) and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.
View details for DOI 10.1016/j.bpj.2013.09.048
View details for Web of Science ID 000327285100020
View details for PubMedID 24268151
Tryptophan (Trp), an intrinsically fluorescent residue of proteins, has been used widely as an energy donor in fluorescence resonance energy transfer (FRET) experiments aimed at measuring intramolecular distances and distance distributions in protein folding-unfolding reactions. However, the high level of heterogeneity associated with the fluorescence lifetime of tryptophan, even in single-tryptophan proteins, imposes restrictions on its use as the energy donor. A search for a tryptophan analogue having reduced lifetime heterogeneity when compared to tryptophan led us to 5-fluorotryptophan (5F-Trp). A single tryptophan-containing mutant form of barstar, a small 89-residue bacterial protein, has multiple lifetime components in its various structural forms including the unfolded state, similar to observations made with several other proteins. Biosynthetic incorporation of 5F-Trp in place of Trp in the mutant barstar resulted in a significant decrease in the level of heterogeneity of fluorescence decay when compared to Trp-barstar, in the native state as well as in the denatured state. Importantly, observation of a major decay component of more than 80% in both the states makes 5F-Trp a significantly better candidate for being the energy donor in FRET experiments, as compared to Trp. This is expected to enable an unambiguous estimation of intramolecular distance distributions during protein folding and unfolding. The sequence insensitivity of the fluorescence decay kinetics of 5F-Trp in proteins was demonstrated by observing the decay kinetics of 5F-Trp incorporated in several synthetic peptides.
View details for DOI 10.1021/jp2016984
View details for Web of Science ID 000291080000034
View details for PubMedID 21574591