The structure of a synthetic peptide comprising the 28 amino-terminal residues of actin has been examined by 1H-NMR and CD spectroscopy. The peptide is largely unstructured and flexible in solution but becomes increasingly structured at higher trifluoroethanol (TFE) concentrations. As judged by CD with the use of two additional peptides (actin 1-20 and actin 18-28), TFE induces formation of up to 48% helical content within residues 1-20, while residues 21-28 exhibit no helical propensity. Similar results were obtained by using NMR-derived distance information in restrained molecular dynamics calculations. The calculated structure of actin 1-28 peptide in 80% TFE is well defined for the first 23 residues with a backbone root mean square deviation of 0.5 A. Two helices are formed from residues 4-13 and 16-20, and a beta-turn is formed from residues 13-16. The N-terminal residues 1-3 exhibit increased flexibility and a helix-like conformation while the C-terminal residues 21-28 show no regular secondary structure. These results are compared with the predicted secondary structure and the structure of the corresponding sequence in the crystal structure of actin [Kabsch et al. (1990) Nature 347, 37-44]. The significance of the TFE-induced peptide structure is discussed.

The flexibility of the polar side chains in the alpha-helical Type I antifreeze protein (AFP) near the solution freezing temperature was investigated by two-dimensional nuclear magnetic resonance spectroscopy. These experiments were conducted to define the rotameric conformations of the proposed ice-binding groups, threonines and asparagines, in order to probe the molecular mechanism for ice binding. On the basis of the 3J alpha beta 2 NMR coupling constant values of 7.1, 8.5, 8.5, and 6.8 Hz for residues T2, T13, T24, and T35, respectively, it can be calculated that the regularly spaced ice-binding threonines sample many possible rotameric states prior to ice binding. The lack of a dominant side chain rotamer is further corroborated by nuclear Overhauser distance measurements for T13 and T24. N16 and N27, both with 3J alpha beta 2 and 3J alpha beta 3 coupling constants of 8.4 and 4.5 Hz, respectively, show a slight preference for the side chain conformation with a chi 1 of -60 degrees. These data suggest that prior to ice binding the threonine and asparagine side chains are free to rotate and that a unique preformed ice-binding structure in solution is not apparent. These observations do not support the rigid side chain model proposed recently by an X-ray study [Sicheri, F., & Yang, D. S. C. (1995) Nature 375, 427-431].

The large paramagnetic shifts and short relaxation times resulting from the presence of ap aramagnetic centre complicate NMR data acquisition and interpretation in solution. As aresult, NMR analysis of paramagnetic complexes is limited in comparison to diamagnetic compounds and often relies on theoretical models.W er eport at oolbox of 1D (1 H, proton-coupled 13 C, selective 1 H-decoupling 13 C, steady-state NOE) and 2D (COSY,NOESY,HMQC) paramagnetic NMR methods that enables unprecedented structural characterisation and in some cases,p rovides more structural information than would be observable for ad iamagnetic analogue.W ed emonstrate the toolboxsb road versatility for fields from coordination chemistry and spin-crossover complexes to supramolecular chemistry through the characterisation of Co II and highspin Fe II mononuclear complexes as well as aC o 4 L 6 cage.