BioNMR

BioNMR (http://www.bionmr.com/forum/)
-   Journal club (http://www.bionmr.com/forum/journal-club-9/)
-   -   Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme (http://www.bionmr.com/forum/journal-club-9/effect-freezing-conditions-distances-their-distributions-derived-double-electron-electron-resonance-deer-study-doubly-spin-labeled-t4-lysozyme-15038/)

nmrlearner 01-25-2012 08:56 AM

Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme
 
Effect of Freezing Conditions on Distances and Their Distributions Derived from Double Electron Electron Resonance (DEER): A Study of Doubly-Spin-Labeled T4 Lysozyme


Publication year: 2012
Source: Journal of Magnetic Resonance, Available online 24 January 2012</br>
Elka R.*Georgieva, Aritro S.*Roy, Vladimir M.*Grigoryants, Petr P.*Borbat, Keith A.*Earle, ...</br>
Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution.We compared the results from rapidly freeze-quenched (?100 ?s) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.</br>
Graphical abstract


http://www.sciencedirect.com/cache/M...000092-fx1.sml</br>Highlights

? DEER with very rapid freezing yielded broader distributions than regular freezing. ? Neither method of freezing altered the distance maxima. ? Rapid freezing yielded more uniform distribution of spin-labels and enhanced DEER S/N. ? Rapid frozen samples require less cryoprotectant than regularly frozen samples. ? Bromo-substituted spin-label produced narrower distance distributions.</br></br>

Source: Journal of Magnetic Resonance


All times are GMT. The time now is 06:12 PM.

Powered by vBulletin® Version 3.7.3
Copyright ©2000 - 2024, Jelsoft Enterprises Ltd.
Search Engine Friendly URLs by vBSEO 3.6.0
Copyright, BioNMR.com, 2003-2013