NMR (Nuclear Magnetic Resonance) is a powerful tool for the structural determination of small proteins in solution at the atomic resolution along with X-ray crystallography for any protein size. It is worth to mention, however,that the protein crystallization is still a problem, namely many proteins are still not readily crystallizable.
On the other hand, monolayer engineering based on Langmuir-Blodgett (LB) method and its modifications is a key technology which yield heat proof, dense and oriented layers of practically all proteins in quasi-cristalline state [1,2]. It would then be very usefull if the structure of the proteins LB films could be determined at atomic resolution by NMR.
Indeed, it is very difficult to apply NMR investigations to LB solid films. The main difficulties are the following. The amount of the material is very small with respect to the weight of the substrate on which it is deposited. Another problem is the rigidity of the LB films. The only one possible technique, which can give results is HR MAS (High Resolution Magic Angle Spinning) NMR spectroscopy [3]. This technique permits to obtain high resolution spectra of the solid samples in the presence of solvent, which increases the mobility of the substance under the investigation.
The aim of the study is to develop a technique of the formation of samples with LB films, suitable for the HR MAS NMR investigations.
LB films of lysosin were deposited onto small crystals of NaCl and Cu(SO4)2. The monolayers of these proteins were formed at the surface of pure water and of condensed salt solutions.
After the depositions, samples were attached to a holder with hole. Water was able to touch the sample through the hole. As the substrate material was water soluble, it was possible to remove some part of it, providing the increase of the sample to substrate weight ratio. According to the estimations, the substrate of 22 mm must have a mass not more than 0.1 mg, in order to be possible to provide NMR measurements. Initially, the supports were more heavy (as it is very difficult to make the deposition onto such small substrates). After the deposition, the additional mass of the support was removed by solving in water. The scheme of the process is demonstrated in the figure.
1. C. Nicolini, Biosensors and Bioelectronics, 10, 105-127
(1995).
2. C. Nicolini, V. Erokhin, F. Antolini, P. Catasti, and P. Facci,
Biochim. Biophys. Acta, 1158, 273-278 (1993).
3. M. Traikia, D.B. Langlais, and P.F. Devaux, J. Magn. Res.,
125, 140 (1997).