Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy |
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Authors: | Anoop M Saxena G Krishnamoorthy Jayant B Udgaonkar and N Periasamy |
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Institution: | (1) Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India;(2) National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore, 565 065, India |
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Abstract: | In protein-folding studies it is often required to differentiate a system with only two-states, namely the native (N) and
unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s)
could also be present. This differentiation of a two-state from a multi-state structural transition is non-trivial when studied
by the several steady-state spectroscopic methods that are popular in protein-folding studies. In contrast to the steady-state
methods, time-resolved fluorescence has the capability to reveal the presence of heterogeneity of structural forms due to
the ‘fingerprint’ nature of fluorescence lifetimes of various forms. In this work, we establish this method by quantitative
analysis of amplitudes associated with fluorescence lifetimes in multiexponential decays. First, we show that we can estimate,
accurately, the relative population of species from two-component mixtures of non-interacting molecules such as fluorescent
dyes, peptides and proteins. Subsequently, we demonstrate, by analysing the amplitudes of fluorescence lifetimes which are
controlled by fluorescence resonance energy transfer (FRET), that the equilibrium folding-unfolding transition of the small
single-domain protein barstar is not a two-step process. |
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Keywords: | Time-resolved fluorescence resonance energy transfer (TR-FRET) fluorescence lifetime two-state protein-folding barstar |
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