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Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated.
The key to the results in our study likely resides in the polarizing nature of the applied force, which selectively stabilizes highly extended unfolded conformations that would be rarely populated in the absence of force.
Trp side chain Hε resonances usually reside above 10 ppm, a region of the spectrum that is rarely populated by resonances from small molecules or peptides (with few exceptions).
Detection and characterisation of rarely populated, partially unfolded states within the native ensemble under physiological conditions, therefore, will be crucial for our understanding of the mechanisms that link folding and aggregation energy landscapes.
Our flow experiments following binding of MutS to GT show that MutS preferably binds in B and then undergoes the transition to U, supporting the suggestion that B is an intermediate in the formation of U. In contrast, MutS appears to preferentially bind a T-bulge in U and remains stably bound with occasional transitions to I, which is also long-lived, and B is rarely populated.
A detailed analysis of conformational states populated in solution using powerful experiments able to detect and analyse the most rarely populated conformations (Table 1), therefore, is crucial for our understanding of the molecular events involved in the initiation of the amyloid cascade.
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MutS GT complexes show relatively rapid transitions between B and U (with large changes in DNA bending), with rare transitions to I. In contrast, the more stable MutS T-bulge coMutS T-bulgevalues of 5 nM for the T-bulge and 40 nM for GT) rarely populate B and undergo infrequent transitions between the long-lived complexesional states, I and U (with only small changes in DNA bending).
This causes their resonances to be shifted to the extreme right region of the spectrum, a region that is rarely (almost never) populated by signals from small peptides or small molecules.
Adjacency is also a social construct, as members of one ethnic group may rarely move to places populated by another ethnic group during a conflict.
Though the show is set mostly on grimy subways and populated by beleaguered characters, rarely a minute goes by onstage without a twinkling eye or a toothy smile.
In this sense, too, the Le Cirque patriarch is in a rarefied club, one populated by other innovative chefs rarely associated with the dishes they popularized, like Jean-Georges Vongerichten, who claims to have created the ubiquitous molten chocolate cake, and Nobu Matsuhisa, who pioneered the pairing of yellowtail fish with jalapeño.
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Since I tried Ludwig back in 2017, I have been constantly using it in both editing and translation. Ever since, I suggest it to my translators at ProSciEditing.

Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com