By Dennis R. Livesay
In Protein Dynamics: tools and Protocols, expert researchers within the box element either experimental and computational how to interrogate molecular point fluctuations. Chapters aspect best-practice recipes protecting either experimental and computational techniques, reflecting smooth protein learn. Written within the hugely successful Methods in Molecular Biology™ series layout, chapters contain introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, with no trouble reproducible laboratory protocols, and key tips about troubleshooting and heading off identified pitfalls.
Authoritative and practical, Protein Dynamics: equipment and Protocols describes the most typical and robust equipment used to symbolize protein dynamics.
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Extra resources for Protein Dynamics: Methods and Protocols
Example text
This B0-dependent dispersion experiment determines a spectral density J(ω) as a function of B0 (¼ ωi/γ i, in which ωi and γ i are resonance angular frequency and the gyromagnetic ratio of the observed nuclear spins, respectively) and has been applied to provide insights into the dynamic processes of biomolecules. However, in this review, we exclusively describe the protocol for the B1-dependent relaxation dispersion for backbone studies, 15 N constant-time (CT) CPMG relaxation (or sometimes called the CPMG R2 dispersion) experiment.
R2 and the uncertainty are numerically calculated using Eqs. 2) and (2) with the peak height ratio and the noise. CPMG Relaxation Dispersion 39 Fig. 4 (a) R2RMSD and (b) χ R202 plot of 15N CPMG dispersion data, calculated using Eq. 4 or 5. The average R2 of each dispersion curve is plotted in (c). Increase in the χ R202 value indicates away from the uniform R2 values. Note that χ R202 may represent the dynamics at the C-terminus well whereas R2RMSD may represent the dynamics in a region between residues 60 and 90 well 5.
After 8 h of growth, inoculate a 50 mL aliquot of M9 growth media with 200 μL of the LB starter culture and incubate at 37 C for 18 h. 6. Use the 50 mL culture to inoculate the remaining 950 mL of M9 growth media. 7. 8. 8. Induce protein expression by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. 9. Allow the protein to express for 4 h at 37 C. 10. Pellet the bacteria by centrifugation and then resuspend in Lysis Buffer. Monitoring Side-Chain Dynamics Using 2H Relaxation 19 11.