25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Atomic resolution characterization of a folding intermediate by pressure-jump NMR

29 Aug 2019, 11:30
25m
Lecture Hall C (Henry Ford Building)

Lecture Hall C

Henry Ford Building

Talk Biological applications Biomolecules

Speaker

Dr Cyril Charlier (NIH)

Description

Understanding how proteins fold into stable structures without external assistance remains one of the major open questions in biophysics. The ability of NMR to report atomic resolution structural information for both folded and unfolded proteins makes it a uniquely powerful to study protein folding. However, typical experiment times of hours to days are incompatible with the typical microsecond to second timescale of folding. Here, we use a new hardware which rapidly and repeatedly switches sample pressure from native (1 bar) to unfolded (2.5 kbar) conditions in less than 3 ms to study the folding of a pressure-sensitized mutant of Ubiquitin. Recently, we demonstrated the presence of two parallel folding pathways where one involves a meta-stable intermediate. Initially, we developed a set of experiments to obtain site-specific 15N and 13C’ chemical shifts of this intermediate state using stroboscopic chemical shift measurement or a reverse-sampled indirect evolution period. In addition, as in this case the intermediate has a relatively long lifetime, we have modified standard 3D experiments to measure the first chemical shift evolution (1H, 15N, 13Ca, 13C’) ~60 ms after the pressure drop, which allows direct observation of cross peaks to intermediate state frequencies. Complemented with other NMR observables such as pressure-jump NOEs and RDCs, we derived a structural model for the intermediate. The model reveals a structure that is similar to the native state of wild type Ubiquitin but but contains multiple non-native H-bonds. This method opens the way for the structural characterization of folding intermediates at atomic resolution.

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