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Transcript
A real example. The rat fatty acid acyl carrier protein. Involved in fatty acid biosynthesis and part of a larger subunit, the synthase, Is it structured by itself?? Summary of the Sequential and Secondary NOEs observed for rat FAS ACP - most definitely structured NHi-NHi+1 iNHi+1 iNHi+1 GDGEAQRDLVKAVAHILGIRDLAGINLDSSLADLGLDSLMGVEVR D D D DD D D D NHi-NHi+2 Hi-NHi+2 Hi-NHi+3 Hi-NHi+4 Hi-Hi+3 CSI J 0-00000---------+-0-0--0+--+0+---+00+-0-----+ ++ -------+--+++++ +++-+++ --- ----- --QILEREHDLVLPIREVRQLTLRKLQEMSSKAGSDTELAAPKSKN NHi-NHi+1 iNHi+1 iNHi+1 D D D D DD D D DDD NHi-NHi+2 Hi-NHi+2 Hi-NHi+3 Hi-NHi+4 Hi-Hi+3 CSI J -----0+-+0++--0--00+--------00000000+0+00-00 -++-+++ - -- -+-+ - -+- +++++++ +++ What next? STRUCTURE CALCULATIONS •From NOE I know close atom-atom distances, but that doesn’t give a structure •The information you have up to this stage is a list of distance constraints •The structure can be determined by inputting this information to computer minimization software. •The computer program also contains information about amino acids, bond lengths/angles and standard information about atom-atom interactions such as minimum distance (i.e. Van der Waals radii) •With all this information you can generate a model of the structure. Important: NMR gives you a number of possible solutions (all almost identical, rmsd <1Å), This can range from 5-20 models X-ray crystallography give one average structure NMR structures can be averaged to give one average structure as well A simulated annealing trajectory over the first few picoseconds 4 helices begin to ‘condense’ Unfolded Correctly folded Representations of 3D Structures C N Precision is not Accuracy These 2D methods work for proteins up to about 100 amino acids, and even here, anything from 50-100 amino acids is difficult. We need to reduce the complexity of these 2D spectra. 1 16 1 H O HN 12 12 C 14 N R2 14 N C 16 O 12 C 12 C 1 R1 1 H HN We can start by replacing 14N with 15N, a spin 1/2 nucleus. HSQC of rat FAS ACP 15N shift of nitrogen of amide bond 1H-15N H N 1H Chemical Shift X 89!
