Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins

Abstract

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on singlemolecule signals detected within 100 s to 1 ms. Distance distributions caused by fluctuations faster than 100 s are studied within these subpopulations by using time-correlated singlephoton counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the ؊1͞2 power (I ؊1/2 ), and that the average contribution to the contour length per base is 0.40 -0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.conformational dynamics ͉ protein folding ͉ single-molecule fluorescence spectroscopy ͉ nucleic acid structure ͉ fluorescence resonance energy transfer I n contrast to neutral homopolymers (1), biopolymers such as proteins and nucleic acids have long-range Coulomb interactions (2) and specific, intrachain interactions that strongly affect their structure and dynamics. Protein folding is the most spectacular manifestation of these interactions (3). Understanding biopolymer energy landscapes requires measurements of fluctuating distance distributions occurring over short distances (0.1-100 nm) and many time scales (picoseconds to minutes) (4). The structure and dynamics of charged polymer chains (polyelectrolytes), including dsDNA and ssDNA, are strongly affected by long-range, electrostatic repulsion (2). Because simulations and theory have focused on dilute, single-chain properties, a regime difficult to access by conventional methods, experimental validation of many predictions has been lacking (5). In protein folding studies, folded, unfolded, and partially folded species may be simultaneously present and rapidly interconverting, obscuring the properties of individual species. An experimental method that unravels distance distributions and fast conformational fluctuations is therefore much-needed.Previous protein folding studies with single-molecule FRET (6-8) measured distance distributions with time resolutions of Ϸ100 s. Using ...

Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy

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Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

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