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Transcript
A b-strand is distinguished by strong CaHi-NHi+1contacts and long range nOes connecting the strands. A long range nOe connects residues more than 5 residues apart in the chain. 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 aiNHi+1 biNHi+1 GDGEAQRDLVKAVAHILGIRDLAGINLDSSLADLGLDSLMGVEVR D D D DD D D D NHi-NHi+2 aHi-NHi+2 aHi-NHi+3 aHi-NHi+4 aHi-bHi+3 CSI J 0-00000---------+-0-0--0+--+0+---+00+-0-----+ ++ -------+--+++++ +++-+++ --- ----- --QILEREHDLVLPIREVRQLTLRKLQEMSSKAGSDTELAAPKSKN NHi-NHi+1 aiNHi+1 biNHi+1 D D D D DD D D DDD NHi-NHi+2 aHi-NHi+2 aHi-NHi+3 aHi-NHi+4 aHi-bHi+3 CSI J -----0+-+0++--0--00+--------00000000+0+00-00 -++-+++ - -- -+-+ - -+- +++++++ +++ So I have assigned the NMR spectrum and connected the amino acids. I have a good idea of the secondary structure. What next?? At this point we notice there are still many nOes we have not assigned on the 2D spectrum. These are neither sequential or short range. They are long-range and connect residues more more than 5 amino acids apart (But still close in space!). O H N CH C O H N CH 2 Asn C O CH C Gly H N H H OH Identified as an asparagine aminohydrogen from COSY spectra HO O C CH 2 Glu H2C C O CH NH 2 NOE indicated the asparagine amino-hydrogen is near a glutamate acidic hydrogen Schematic showing long range nOes in the lac headpiece protein 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 The power of the NOESY experiment is that the intensity of an NOE peak will be related to the nuclear separation. Strong NOE crosspeaks - 1.8-2.5 Å Medium NOE crosspeaks – 1.8-3.5 Å Weak NOE crosspeaks – 1.8-5.0Å Excerpt from an NOE table for Actinorhodin Polyketide ACP - 1997 This file contained ~ 700 lines of nOe restraints ! Thr7 NH assign (resid 7 and name HN )(resid 75 and name HD1* ) 4.0 2.2 0.5 assign (resid 7 and name HN )(resid 75 and name HD2* ) 4.0 2.2 0.5 assign (resid 10 and name HN )(resid 75 and name HD2* ) 5.0 3.2 0.5 assign (resid 10 and name HN )(resid 75 and name HD1* ) 3.3 1.5 1.0 assign (resid 72 and name HN )(resid 31 and name HD1* ) 5.0 5.0 0.5 assign (resid 72 and name HN )(resid 31 and name HD2* ) 3.3 1.5 0.5 assign (resid 72 and name HN )(resid 31 and name HB* ) 4.0 4.0 1.5 assign (resid 72 and name HN )(resid 31 and name HA ) 4.0 4.0 1.0 75 and name HN )(resid 10 and name HD1* ) 4.5 4.5 1.0 ! Leu10 NH !Arg72 NH ! Leu 75 NH assign (resid The simulated annealing protocol. Begin by simulating a 1000K heat bath and generate an extended model strand along x (random coordinates along z,y) Start x Apply the distance restraints from the NOE data (perhaps 1000 restraints for a protein of 90 amino acids). Weight the nOes to favour the formation of local secondary structure and later long range structure. Allow chain to move through itself by reducing the effective Van Der Waals radii 30 ps Start to cool the system and increase the penalty for not satisfying an NOE. 20 ps Minimize the final structure to see if it satisfies all the nOes A simulated annealing trajectory over the first few picoseconds 4 helices begin to ‘condense’ Unfolded Correctly folded Challenges for Interpreting 3D Structures • To correctly represent a structure (not a model), the uncertainty in each atomic coordinate must be shown • Polypeptides are dynamic and therefore occupy more than one conformation – Which is the biologically relevant one? Representation of Structure Conformational Ensemble Neither crystal nor solution structures can be properly represented by a single conformation Intrinsic motions Imperfect data Uncertainty RMSD of the ensemble 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 Ha O HN 12 12 C 14 N R2 14 N Ca 16 O 12 C 12 Ca 1 R1 1 Ha 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!