Proton nmr relaxation study of the dynamics of anthopleurin‐A in solution

Abstract

Spin-spin and spin-lattice 1H-nmr relaxation times of the sea anemone polypeptide anthopleurin-A were measured at frequencies of 200, 300, 400, and 500 MHz. Relaxation times were fitted iteratively by least squares regression to the isotropic tumbling model, Woessner's model for anisotropic motion, and Lipari and Szabo's "model-independent" model. Data for aromatic and aliphatic methine protons could not be fitted satisfactority using the isotropic model. Good fits were obtained, however, using the model-independent approach, indicating that high-frequency internal motions of the polypeptide backbone were significant. In addition, a range of tau c values from 2.2 to 3.2 ns was obtained for various methine protons, suggesting that overall rotational reorientation of the molecule was anisotropic. Methyl group relaxation data were fitted satisfactorily by Woessner's model. Some assessment has been made of the effect of experimental errors on the quality of fit to the data, as well as of the contribution of experimental values at certain frequencies to definition of the spectral density function.

Site-3 toxins and cardiac sodium channels

“…Structural information is available for several of the site-3 toxins, including a 1.2 Å crystal structure for the α-scorpion toxin from Androctonus australis (Housset et al, 1994). NMR coordinates for Ap-A and Ap-B, although determined and reported earlier, were released in 1996 (Torda & Norton, 1989;Scanlon & Norton, 1994). For more information see Smith and Blumenthal in this issue.…”

Helical Molecular Springs with Varying Spring Constants

“…To measure the flexibility of the two different helices, we employed the classical Lipari‐Szabo model, [10] which is commonly used to determine the flexibility of different regions in proteins [12] . With the Lipari‐Szabo model‐free analysis, a general expression can be derived, which links the frequency‐dependence of the longitudinal relaxation time ( T 1 ) to the effective correlation time for internal motions ( τ e ) and the generalized order parameter squared ( δ 2 ) for specific proton resonances [10c] . To apply the Lipari‐Szabo model to our shape‐defined ladder polymers, we first measured the T 1 relaxation times of the average H f proton resonances in the polymers at different Larmor frequencies (300 MHz, 400 MHz, 500 MHz, 600 MHz, and 800 MHz) with inversion‐recovery 1 H NMR pulse sequences (see Figures 4a and b for example spectra).…”

“…To apply the Lipari‐Szabo model to our shape‐defined ladder polymers, we first measured the T 1 relaxation times of the average H f proton resonances in the polymers at different Larmor frequencies (300 MHz, 400 MHz, 500 MHz, 600 MHz, and 800 MHz) with inversion‐recovery 1 H NMR pulse sequences (see Figures 4a and b for example spectra). Next, we fit all the T 1 relaxation data at the different Larmor frequencies with the model‐free approach of Lipari and Szabo [10a, b] (as detailed in the report of Torda and Norton for proton NMR spectra [10c] ), which afforded (Figures 4c and d) the effective correlation times for internal motions ( τ e ) and the generalized order parameters squared ( δ 2 ) for the average proton resonances H f in both polymers. Notably, for syn ‐helix 4 , δ 2 (which ranges from 0 to 1.0, with 0 being the most flexible) is significantly lower than for anti ‐helix 5 , which directly confirms the increased flexibility of the helix with the larger diameter experimentally, as predicted by our MD simulations (Figures 3 and S5) and the force constants (Figure 1) determined from the minimum energy coordinate scans.…”

NMR Structure Determination for Oligonucleotides

“…Non-conforming RDC restraints and HSQC cross peak ambiguities can also be indicators of slower time-frame dynamics (Rossetti et al, 2015;Smrcka et al, 2010). Two methods to explore conformational space whilst still using NMR restraints are time-averaged restraints and ensemble or replica averaged restraints (Cavalli, Camilloni, & Vendruscolo, 2013;Torda & Norton, 1989). Here, we describe the implementation of MDtar (time-averaged restraints) in Amber.…”