Kinetics of chain motions within a protein-folding intermediate

Abstract

Small proteins can fold remarkably rapidly, even in μs. What limits their rate of folding? The Engrailed homeodomain is a particularly well-characterized example, which folds ultrafast via an intermediate, I, of solved structure. It is a puzzle that the helix2-turn-helix3 motif of the 3-helix bundle forms in approximately 2 μs, but the final docking of preformed helix1 in I requires approximately 20 μs. Simulation and structural data suggest that nonnative interactions may slow down helix docking. Here we report the direct measurement of chain motions in I by using photoinduced electron transfer fluorescence-quenching correlation spectroscopy (PET-FCS). We use a mutant that traps I at physiological ionic strength but refolds at higher ionic strength. A single Trp in helix3 quenches the fluorescence of an extrinsic label on contact with it. We placed the label along the sequence to probe segmental chain motions. At high ionic strength, we found two relaxations for all probed positions on the 2-and 20-μs time scale, corresponding to the known folding processes, and a 200-ns phase attributable to loop closure kinetics in the unfolded state. At low ionic strength, we found only the 2-μs and 200-ns phase for labels in the helix2-turn-helix3 motif of I, because the native state is not significantly populated. But for labels in helix1 we observed an additional approximately 10-μs phase showing that it was moving slowly, with a rate constant similar to that for overall folding under native conditions. Folding was rate-limited by chain motions on a rough energy surface where nonnative interactions constrain motion.

Intrinsic Motions in the N-Terminal Domain of an Ionotropic Glutamate Receptor Detected by Fluorescence Correlation Spectroscopy

“…Oxazine fluorophores are advantageous for such studies because they lack photophysical fluorescence fluctuations at moderate excitation energies and facilitate the detection of protein chain motions at the single-molecule level with high signal-to-noise ratio. 28 The recorded ACFs fitted well to a model for a monodisperse molecular diffusion without any additional fluorescence fluctuations in the submillisecond time domain, in agreement with rigidbody globules (Fig. 2b).…”

“…26,27 Recently, kinetics of helix motions have been uncovered successfully using this technique. 28 We searched for motions along specific conformational coordinates in the AMPAR NTD using PET fluorescence quenching. Native Trp side chains (positions 124, 152, 256, 317, and 368) were structurally remote from the introduced labeling sites of the fluorophore (N163C and D225C; Fig.…”

“…In combination with fluorescence correlation spectroscopy (FCS), the method is being used to detect kinetics of protein structural fluctuations at the level of individual molecules on timescales from nanoseconds (ns) to milliseconds (ms). [26][27][28] Here, we used this technique to search for motions within the AMPAR GluA2 NTD. We identified sitespecific sub-millisecond protein dynamics.…”

Malleability of folding intermediates in the homeodomain superfamily

“…Temperature-jump experiments using IR to monitor secondary structure formation and Trp fluorescence for tertiary interactions on wild-type protein and the sequence of residues 16-59 that encompass the HTH domain show conclusively that the 1.7-μs phase is for the formation of the HTH domain with concerted consolidation of its secondary and tertiary structures (7). Direct measurements of the motion of H1 by photoinduced electron transfer fluorescence-quenching correlation spectroscopy shows that the slower step of 17 μs represents the segmental motion of H1 as it docks onto the HTH motif (11). The folding is sufficiently fast that full atomistic MD simulations have been performed of the protein on similar time scales to those of experiment to fill in the folding pathway (4,12).…”

Folding of the Pit1 homeodomain near the speed limit

“…The H2-turn-H3 domain forms both secondary and tertiary structure in 1.7 μs, and the docking of H1 takes 17 μs. Direct measurements of the motion H1 by photoinduced electron transfer fluorescence-quenching correlation spectroscopy shows that 17 μs is close to the time constant of segmental motion of H1 (6). The folding is sufficiently fast that full atomistic molecular dynamics simulations have been done on the protein on similar time scales to those of experiment to fill in the folding pathway (7,8).…”