Nuclear magnetic resonance protein system

The use of computer simulations to study internal motions and thermodynamic properties is receiving increased attention. One important use of the method is to provide a more fundamental understanding of the molecular information contained in various kinds of experiments on these complex systems. In the first part of this paper we review recent work in our laboratory concerned with the use of computer simulations for the interpretation of experimental probes of molecular structure and dynamics of proteins and nucleic acids. The interplay between computer simulations and three experimental techniques is emphasized (1) nuclear magnetic resonance relaxation spectroscopy, (2) refinement of macro-molecular x-ray structures, and (3) vibrational spectroscopy. The treatment of solvent effects in biopolymer simulations is a difficult problem. It is not possible to study systematically the effect of solvent conditions, e.g. added salt concentration, on biopolymer properties by means of simulations alone. In the last part of the paper we review a more analytical approach we have developed to study polyelectrolyte properties of solvated biopolymers. The results are compared with computer simulations. 

McMahon, M. T., deDios, A. D, Godbout, N., Salzmann, R., Laws, D. D., Le, H., Havlin, R. H., Oldfield, E., 1998, An Experimental and Quantum Chemical Investigation of CO Binding to Heme Proteins and Model Systems A Unified Model Based on 13C, 170 and 57Fe Nuclear Magnetic Resonance and 57Fe Mossbauer and Infrared Spectroscopies , J. Am. Chem. Soc., 120, 4784. 

Response to external field is used to determine magnetic susceptibility and electronic ground state of spin system Nuclear magnetic resonance (NMR) sjjectra Observe some atoms from protein and solvent, report on protein and, indirectly, the iron center. 

One example of this possible existence of a hierarchy of receptor states has been discussed by Frauenfelder (1988). He reviewed studies on the binding of substrates and ligands to myoglobin. The process follows a power law, characterizing the protein as a complex system. Nuclear magnetic resonance (NMR) analyses revealed a number of conformational substates. 

Fig. 8. Heteronuclear single-quantum coherenc (HSQC) spectrum of the hypothetical protein of the flowering locus T protein produced in the cell-free system. The FT protein was synthesized in the same way as in Fig. 6 except that Ala, Leu, Gly, and Gin in both translation and substrate mixture were replaced with their -labeled forms (Isotec, Inc ). After incubation for 48 h, the reaction mixture (1 mL) was dialyzed against 10 mMphosphate buffer (pH 6.5) overnight, and then centrifuged at 30,000g for 10 min. The supernatant containing 30 xMof the protein was directly subjected to nuclear magnetic resonance spectroscopy. The spectrum was recorded on a Broker DMX-500 spectrometer at 25°C, and 2048 scans were averaged for the final H- WHSQC spectrum.

This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of I I-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification. 

The main constituents of cell membranes are lipids and proteins. Therefore, we have to study possible associations between these macromolecules and molecules having anesthetic potency. It is instructive, however, at a preliminary stage, to study simpler systems which exhibit some of the basic patterns of asociation that occur in membranes. The results of such studies will be described and discussed in the present chapter. The method used has been mainly infrared spectroscopy. While other techniques, like Raman and nuclear magnetic resonance spectroscopy turn out to be more powerful tools in many cases, infrared spectra can be highly informative in others, especially when polar groups are involved. 

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