Structural Dynamics of Erabutoxin b

Abstract

A detailed examination has been made of tlie I3C NMR relaxation times of the assigned methyl groups of erabutoxin b. Anisotropic rotation was analysed using a restricted diffusion model. The results are compared with a previous study of the 'H NMR relaxation times. There is good agreement on the segments of the molecule which show restricted motion. Our results are compared with those of previous studies on proteins in solution and in crystals and again tlie general agreement is good. We have attempted to interpret the value of the limited motion seen in tlie protein to the reactions of the neurotoxin.Much experimental evidence shows that there is considerable internal motion in proteins in solution [I -31 (and references cited in [3]), in agreement with theoretical treatments of protein dynamics which indicate that there are internal motion in the 1 -100-ps range [4,5]. The idea that a protein has a dynamic flexible structure would seem to be especially important for the rate of protein-protein interaction where two complicated surfaces must come together. We have pointed out that a study of neurotoxins which bind to membrane receptor proteins could provide a good test case. We have therefore chosen to examine erabutoxin b, a small protein with 62 amino acids, in detail by NMR methods in order to see if molecular motion is important for its binding steps to the acetylcholine receptor protein. We started this work using proton NMR and, in a previous paper, we have reported the analysis of the relaxation behaviour of methyl protons in erabutoxin b [6]. There is a methodological difficulty, however, in that tlie contribution of external protons to the relaxation of methyl groups has to be analysed in addition to the dynamics of the methyl groups themselves. Despite these difficulties, the 'H relaxation study generated considerable knowledge about the motion of the methyl groups in the protein. In contrast, I3C relaxation times are dominantly determined by the nearest proton(s) and interpretation of relaxation in terms of molecular motions is then more direct [7]. In the present study, we report the relaxation behaviour of the methyl carbons in erabutoxin b and elucidate tlie segmental motion of its side chains. Unfortunately these 13C NMR studies have had to be made in non-dilute solutions which could affect relaxation processes of surface residues. Thus a comparison with tlie proton NMR observation is very valuable. It will be shown here that the two studies of methyl group motion in erabutoxin b give very similar results. We can then comment on the possible functional importance of these motions. EXPERIMENTAL PROCEDUREErabutoxin b was isolated from tlie venom of Laticauda .scwz@i.sc.iti/u as described previously [8]. After purification, Ahhrrviutions. T I , spin-lattice relaxation time; NOE, nuclear Overhauser effect.-~ it was lyophilized from 0.1 M acetic acid solution. For NMR measurements, samples of erabutoxin b were dissolved in 99.5 % 2H20 to a concentration of 20 mM. The pH of the samples was adjusted to ...

Methyl motions in 13C-methylated concanavalin as studied by carbon-13 magnetic resonance relaxation techniques

“…The range of DMc frequencies given in Table III is similar to those determined for other protein side chain methyl groups from NMR data using the wobble in a cone or restricted diffusion models (Blakley et al, 1978;Wittebort et al, 1979;Richarz et al, 1980;Inagaki et al, 1982).…”

“…Next, a calculation was performed which allowed two free diffusion internal rotations in addition to the overall isotropic motion of the protein. Z)Mc was varied from 1 X 1010 to 5 X 1010 s"', typical for methyl groups (Blakley et al, 1978;Richarz et al, 1980;Wittebort et al, 1979;Jones et al, 1976;Inagaki et al, 1982), and Z))m, representing free diffusion about the C'-N* or C"-Na bonds, was varied from 109 to 1012 s'1.…”

An electrostatic model of a membrane ion pump

“…E ven w ithin a solid single crystal of a com pact protein, X -ray diffraction reveals typical root m ean square am plitudes of m otion of + 0.05 nm for th e m ain backbone chain atom s and + 0.1 nm for some side chain atom s (Frauenfelder et al 1979;A rtym ink et al 1979). N uclear m agnetic resonance of proteins in solution reveals th a t com plete loops of secondary stru ctu re m ay undergo segm ental m otion of large am plitude (Inagaki et al 1982).…”