Water-protein interactions play a direct role in protein folding. The chain collapse that accompanies protein folding involves extrusion of water from the nonpolar core. For many proteins, including apomyoglobin (apoMb), hydrophobic interactions drive an initial collapse to an intermediate state before folding to the final structure. However, the debate continues as to whether the core of the collapsed intermediate state is hydrated and, if so, what the dynamic nature of this water is. A key challenge is that protein hydration dynamics is significantly heterogeneous, yet suitable experimental techniques for measuring hydration dynamics with site-specificity are lacking. Here, we introduce Overhauser dynamic nuclear polarization at 0.35 T via site-specific nitroxide spin labels as a unique tool to probe internal and surface protein hydration dynamics with site-specific resolution in the molten globular, native, and unfolded protein states. The 1H NMR signal enhancement of water carries information about the local dynamics of the solvent within ~10 Å of a spin label. EPR is used synergistically to gain insights on local polarity and mobility of the spin-labeled protein. Several buried and solvent-exposed sites of apoMb are examined, each bearing a covalently bound nitroxide spin label. We find that the hydrophobic core of the apoMb molten globule is hydrated with water bearing significant translational dynamics, only 4–6-fold slower than that of bulk water. The hydration dynamics of the native state is heterogeneous, while the acid-unfolded state bears fast-diffusing hydration water. This study provides a high-resolution glimpse at the folding-dependent nature of protein hydration dynamics.
Myoglobins are ubiquitous proteins that play a seminal role in oxygen storage, transport, and NO metabolism. The folding mechanism of apomyoglobins from different species has been studied to a fair extent over the last two decades. However, integrated investigations of the entire process, including both the early (sub-ms) and late (ms-s) folding stages, have been missing. Here, we study the folding kinetics of the single-Trp Escherichia coli globin apoHmpH via a combination of continuous-flow microfluidic and stopped-flow approaches. A rich series of molecular events emerges, spanning a very wide temporal range covering more than 7 orders of magnitude, from sub-microseconds to tens of seconds. Variations in fluorescence intensity and spectral shifts reveal that the protein region around Trp120 undergoes a fast collapse within the 8 μs mixing time and gradually reaches a native-like conformation with a half-life of 144 μs from refolding initiation. There are no further fluorescence changes beyond ca. 800 μs, and folding proceeds much more slowly, up to 20 s, with acquisition of the missing helicity (ca. 30%), long after consolidation of core compaction. The picture that emerges is a gradual acquisition of native structure on a free-energy landscape with few large barriers. Interestingly, the single tryptophan, which lies within the main folding core of globins, senses some local structural consolidation events after establishment of native-like core polarity (i.e., likely after core dedydration). In all, this work highlights how the main core of the globin fold is capable of becoming fully native efficiently, on the sub-millisecond time scale.
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