The proteins are essential actors of all the processes of the alive one. For their operation, these chains of amino acids must form a well defined structure, and must thus “be folded up” correctly. The incorrect folding up of certain proteins is in the beginning various pathologies like the neurodegenerative diseases of Alzheimer and Parkinson, or the diseases implying the protein prior (insane cow, Kreutzfeld-Jacob). The knowledge of the fundamental mechanisms of the folding up and the stability of proteins, as well as kinetics of inter conversion from one state to another, thus remains a major objective in the comprehension of the molecular bases of the alive one. The spectroscopy by Nuclear Magnetic Resonance (NMR) multidimensional is the method of choice to study the structural and dynamic protein properties in solution. But the study of kinetic processes like folding up or the unfolding of proteins by this technique is limited by important times of measurement which characterize it. At the Laboratory of Magnetic Resonance Nuclear at the Institute of Structural Biology (IBS-LRMN), we succeeded in reducing times of measurement of two-dimensional spectra NMR of a few minutes to a few seconds by combining technological projections (cryogenic probes, injecting rapid) with spectroscopic developments (experiments of fast NMR). This new technique, SOFAST 2D NMR, enable us to follow the folding up or unfolding of a protein in real time with a temporal resolution of about a second simultaneously for a great number of atoms along the peptide chain.

 Figure-1:SOFAST 2D NMR: A technique to follow molecular kinetics in real time 

In collaboration with the chemistry laboratory and Biology of Metals of the Institute of Research in Technologies and Sciences for the alive one, this new technique was applied to the observation in real time of the transition from the protein &-lactalbumine of a state “molten globule” towards the native state. The observation identical speeds of folding up along the peptide chain confirms that this transition is controlled by only one state from transition.

Figure 2: Kinetics of folding up of the protein a-lactalbumine measured by SOFAST 2D NMR

 In a second application, the researchers characterized the spontaneous unfolding of the protein ubiquities under native conditions. Measurement in real time of times characteristic of hydrogen exchange - deuterium for the groupings amides with high pH gives access at the speeds of local unfolding along the peptide chain. The data show that the kinetics of unfolding are not uniform through protein, and the researchers in particular observed a gradual increase speeds of unfolding through the layer.

 This new technique, SOFAST 2D NMR, are applicable to the kinetic studies of process like folding up or unfolding, certain enzymatic reactions, or of the chemical exchanges for any protein having a molecular mass lower than approximately 20 kDa. Combined with recent projections in the field of “the NMR in cell”, this approach should also make it  possible to characterize molecular kinetics directly in a cellular environment. More: Protein folding camera and unfolding studied At atomic resolution by fast two-dimensional NMR spectroscopy Paul Schanda, Vincent Forges & Bernhard Brutscher Proc. Natl. Acad. Sc the USA (2007), 104: 11257-11262

Figure 3: Measure speeds of hydrogen-deuterium exchange in the protein ubiquitine with pH 12 reflecting speeds of local unfolding through the peptide chain.