Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( k e x , F I 1 ~ 550 s-1, k e x , F I 2 ~ 1200 s-1, k e x , I 1 I 2 ~ 5000 s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15 °C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

A CEST NMR experiment to obtain glycine 1Hα chemical shifts in 'invisible' minor states of proteins.

Chemical exchange saturation transfer (CEST) experiments are routinely used to study protein conformational exchange between a 'visible' major state and 'invisible' minor states because they can detect minor states with lifetimes varying from ~ 3 to ~ 100 ms populated to just ~ 0.5%. Consequently several 1H, 15N and 13C CEST experiments have been developed to study exchange and obtain minor state chemical shifts at almost all backbone and sidechain sites in proteins. Conspicuously missing from this extensive set of CEST experiments is a 1H CEST experiment to study exchange at glycine (Gly) 1Hα sites as the existing 1H CEST experiments that have been designed to study dynamics in amide 1H-15N spin systems and methyl 13CH3 groups with three equivalent protons while suppressing 1H-1H NOE induced dips are not suitable for studying exchange in methylene 13CH2 groups with inequivalent protons. Here a Gly 1Hα CEST experiment to obtain the minor state Gly 1Hα chemical shifts is presented. The utility of this experiment is demonstrated on the L99A cavity mutant of T4 Lysozyme (T4L L99A) that undergoes conformational exchange between two compact conformers. The CEST derived minor state Gly 1Hα chemical shifts of T4L L99A are in agreement with those obtained previously using CPMG techniques. The experimental strategy presented here can also be used to obtain methylene proton minor state chemical shifts from protein sidechain and nucleic acid backbone sites.

Revisiting the Woolly wolf (Canis lupus chanco) phylogeny in Himalaya: Addressing taxonomy, spatial extent and distribution of an ancient lineage in Asia.

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.

A Double-Resonance CEST Experiment To Study Multistate Protein Conformational Exchange: An Application to Protein Folding.

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.

Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange.

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a 'visible' major state and 'invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~ 50 °C) that are sometimes required to populate protein minor conformers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to 'dips' in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18 kDa T4 Lysozyme (T4L) at 50 °C and the ~ 11 kDa E. coli hibernation promoting factor (HPF) at 52 °C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~ 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.