The 2 H, 13 C, 15 N-labeled, 148-residue integral membrane protein OmpX from Escherichia coli was reconstituted with dihexanoyl phosphatidylcholine (DHPC) in mixed micelles of molecular mass of about 60 kDa. Transverse relaxation-optimized spectroscopy (TROSY)-type triple resonance NMR experiments and TROSY-type nuclear Overhauser enhancement spectra were recorded in 2 mM aqueous solutions of these mixed micelles at pH 6.8 and 30°C. Complete sequence-specific NMR assignments for the polypeptide backbone thus have been obtained. The 13 C chemical shifts and the nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX͞DHPC in solution and in the collection of an input of conformational constraints for the computation of the global fold of the protein.The same type of polypeptide backbone fold is observed in the presently determined solution structure and the previously reported crystal structure of OmpX determined in the presence of the detergent n-octyltetraoxyethylene. Further structure refinement will have to rely on the additional resonance assignment of partially or fully protonated amino acid side chains, but the present data already demonstrate that relaxation-optimized NMR techniques open novel avenues for studies of structure and function of integral membrane proteins.A bout one-third of the genes in living organisms are assumed to encode integral membrane proteins (e.g., refs. 1-3), and three-dimensional (3D) structure determination of this class of proteins is fundamental to the understanding of a wide spectrum of biological functions. Notwithstanding the crucial importance of work in this area, the database of 3D membrane protein structures is still small, which reflects the challenge presented by this class of molecules to structural biologists. In particular, the solution NMR techniques that commonly are applied with biological macromolecules (e.g., refs. 4 and 5) so far only in few instances have been used with membrane proteins (e.g., refs. 6 and 7) or membrane-binding polypeptides (e.g., refs. 8 and 9), whereby appropriate detergents were used to keep the proteins in solution. Suitable micelles for such studies must ensure the structural and functional integrity of the membrane protein, should be a good mimic of the natural environment in the cell membrane, and need to be sufficiently small to allow rapid Brownian motions of the mixed micelles in solution (e.g., refs. 10-12). The large size of the structures obtained upon reconstitution and solubilization of membrane proteins in detergent micelles actually has limited the application of solution NMR techniques to such systems because of the slow tumbling in solution and the concomitantly large linewidths. New NMR techniques are now available to extend the size limits for NMR in solution, i.e., transverse relaxation-optimized spectroscopy (TROSY) (13) and cross-correlated relaxation-enhanced polarization transfer (CRINEPT) (14), and high-quality NMR spectra have been presented for protei...

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation-optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY-based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence-specific NMR assignments have been obtained for the polypeptide backbone. The 13 C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA L L-barrel from those of OmpX. Overall, the present data demonstrate that relaxationoptimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins. ß

Abstract:In this paper attenuated total reflectance Fourier transform infrared spectroscopy has been employed for the study of the structural composition of aggregates of the oligomeric L-asparaginase II from E.coli formed in the presence of glycerol after the induction of refolding of the protein. Apart from the perfect coincidence of the secondary structure composition of EcA2 as determined by FTIR and crystallography [1], it has also been shown that secondary structure of protein in asparaginase deposits is similar to that of the native conformation: 20.7% extended, 22.3% disordered, 31.4% helix and 25.6% turn/bend/β sheet. Certain structural similarities in the range of experimental error was observed for all three protein deposits presented in this paper, indicating a common structural basis for the composition of this types of aggregates. It is concluded that in the constitution of such precipitates, a partially folded (molten globule like) state(s) is involved, and its stabilisation is a key factor leading to the aggregation. The results presented in this paper might serve to be a good explanation and an excellent basis for the fundamental theory of protein (oligomers) precipitation by osmotic substances.Keywords: attenuated total reflectance Fourier transform infrared spectroscopy, the oligomeric L-asparaginase II, secondary structure Abbreviations: EcA2[WT] -E.coli L-asparaginase II wild type. ATR-FTIR -attenuated total reflectance Fourier transform infrared spectroscopy. MW -molecular weight. PEGpoly(ethylene glycol). IRE -internal reflection element.

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