User:Calhobbs11/Protein folding

Protein Nuclear Magnetic Resonance (NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies. Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s. Some of the main techniques for studying proteins structure and non-folding protein structural changes include COSY, TOCSY,  HSQC, Time relaxation (T1 & T2), and NOE. NOE is especially useful because as it perturbs hydrogens through magnetization, exchanges between spatially proximal hydrogens are observed.

Because Protein folding takes place in about 50 to 3000 s−1 CPMG Relaxation dispersion and Chemical exchange Saturation Transfer have become some of the primary techniques for NMR analysis of folding. In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape. To do this, CPMG Relaxation dispersion takes advantage of the Spin echo phenomenon which exposes the target nuclei to a 90 pulse followed by one or more 180 pulses. As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the the thermodynamics and kinetics between the excited and ground. Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state. This signal is amplified by decreasing the magnetization (and the signal) of the ground state.

The main limitations in NMR is that its resolution decreases sensitivity with proteins that are larger than 25 kDa and in general is not as detailed as X-ray crystallography. Additionally, Protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum.

In a study focused on the folding of an Amyotrophic lateral sclerosis involved protein SOD1, excited intermediates were studied with Relaxation dispersion and Saturation transfer. SOD1 had been previously tied to many disease causing mutants which were supposed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding of SOD1 mutants.