Conformational dynamics play critical roles in protein folding, misfolding, function, misfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FESs) remain a challenge, NMR spectroscopy has emerged as an invaluable experimental tool to explore the FES of a protein, as conformational dynamics can be probed at atomic resolution over a wide range of timescales. Here, we use chemical exchange saturation transfer (CEST) to detect “invisible” minor states on the energy landscape of the A39G mutant FF domain that exhibited “two-state” folding kinetics in traditional experiments. Although CEST has mostly been limited to studies of processes with rates between ∼5 to 300 s−1 involving sparse states with populations as low as ∼1%, we show that the line broadening that is often associated with minor state dips in CEST profiles can be exploited to inform on additional conformers, with lifetimes an order of magnitude shorter and populations close to 10-fold smaller than what typically is characterized. Our analysis of CEST profiles that exploits the minor state linewidths of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverting states spanning over two orders of magnitude in timescale from ∼100 to ∼15,000 μs. A similar folding scheme is established for the wild-type domain as well. The study shows that the folding of this small domain proceeds through a pair of sparse, partially structured intermediates via two discrete pathways on a volcano-shaped FES.
Of the sub-species of Holarctic wolf, the Woolly wolf (Canis lupus chanco) is uniquely adapted to atmospheric hypoxia and widely distributed across the Himalaya, Qinghai Tibetan Plateau (QTP) and Mongolia. Taxonomic ambiguity still exists for this sub-species because of complex evolutionary history anduse of limited wild samples across its range in Himalaya. We document for the first time population genetic structure and taxonomic affinity of the wolves across western and eastern Himalayan regions from samples collected from the wild (n = 19) using mitochondrial control region (225bp). We found two haplotypes in our data, one widely distributed in the Himalaya that was shared with QTP and the other confined to Himachal Pradesh and Uttarakhand in the western Himalaya, India. After combining our data withpublished sequences (n = 83), we observed 15 haplotypes. Some of these were shared among different locations from India to QTP and a few were private to geographic locations. A phylogenetic tree indicated that Woolly wolves from India, Nepal, QTP and Mongolia are basal to other wolves with shallow divergence (K2P; 0.000-0.044) and high bootstrap values. Demographic analyses based on mismatch distribution and Bayesian skyline plots (BSP) suggested a stable population over a long time (~million years) with signs of recent declines. Regional dominance of private haplotypes across its distribution range may indicate allopatric divergence. This may be due to differences in habitat characteristics, availability of different wild prey species and differential deglaciation within the range of the Woolly wolf during historic time. Presence of basal and shallow divergence within-clade along with unique ecological requirements and adaptation to hypoxia, the Woolly wolf of Himalaya, QTP, and Mongolian regions may be considered as a distinct an Evolutionary Significant Unit (ESU). Identifying management units (MUs) is needed within its distribution range using harmonized multiple genetic data for effective conservation planning.
Despite
the importance of protein dynamics to function, studying
exchange between multiple conformational states remains a challenge
because sparsely populated states are invisible to conventional techniques.
CEST NMR experiments can detect minor states with lifetimes between
5 and 200 ms populated to a level of just ∼1%. However, CEST
often cannot provide the exchange mechanism for processes involving
three or more states, leaving the role of the detected minor states
unknown. Here a double-resonance CEST experiment to determine the
kinetics of multistate exchange is presented. The approach that involves
irradiating resonances from two minor states simultaneously is used
to study the exchange of T4 lysozyme (T4L) between the dominant native
state and two minor states, the unfolded state and a second minor
state (B), each populated to only ∼4%. Regular CEST does not
provide the folding mechanism, but double-resonance CEST clearly shows
that T4L can fold directly without going through B.
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