Solving NMR structures Part I: Commonly used experimentally derived restraintsUsing NOESY to generate nOe distance restraintsnOe buildup in NOESYspin diffusionCrosspeaks due to spin diffusion exhibit delayed buildup in NOESY experimentsOther nOe caveatsThe goal: translating NOESY crosspeak intensities into nOe distance restraintsCalibration of nOe’sCoupling constants and dihedral anglesEmpirical Karplus relations in proteinsEmpirical Karplus relations in proteinsMeasuring 3JHN-Ha: 3D HNHA spectra3D HNHB experiment3D HN(CO)HB experimentHNHB and HN(CO)HB togetherHNHB, HN(CO)HB togetherDihedral angle restraintsAmide hydrogen exchangeSlide 19Protection factorsMeasuring amide exchange ratesPowerPoint PresentationSlide 23Hydrogen bond restraintsSolving NMR structures Part I:Commonly used experimentally derived restraintsDistance restraints from crosspeak intensities in NOESY spectra; measuring and calibrating NOEsDihedral angle restraints from three-bond J couplings; measuring J couplingsHydrogen bond restraints from amide hydrogen exchange protection dataUsing NOESY to generate nOe distance restraints•NOESY measurements are not steady-state nOe’s: we are not saturating one resonance with constant irradiation while observing the effects at another. •Instead, we are pulsing all of the resonances, and then allowing nOe’s to build up through cross-relaxation during a mixing time --so nOe’s in a NOESY are kinetic: crosspeak intensities will vary with mixing time•typical tm’s used in an NOESY will be 20-200 ms.fromGlasel &Deutscherp. 354basic NOESY pulse sequencemixing timenOe buildup in NOESY•other things being equal, the initial rate of buildup of a NOESY crosspeak is proportional to 1/r6, where r is the distance between the two nuclei undergoing cross-relaxation. •nOe buildup will be faster for larger proteins, which have a longer correlation time tc, and therefore more efficient zero-quantum cross-relaxation•initially crosspeak intensity builds up linearly with time, but then levels off, and at very long mixing time will actually start to drop due to direct (not cross) relaxation.spin diffusion•under certain circumstances, indirect cross-relaxation pathways can be more efficient than direct ones, i.e. A to B to C more efficient than A to C. This is called spin diffusion•when this happens the crosspeak intensity may not be a faithful reflection of the distance between the two nuclei.Crosspeaks due to spin diffusion exhibit delayed buildup in NOESY experiments-101234560 50 100 150 200 250 300relative crosspeak intensitymixing time spin diffusiondirect cross-relaxationnote the delay in buildup•spin diffusion peaksare usually observedat long mixing time, and their intensity does not reflect the initial rate of buildup•these effects can be avoided either by sticking with short mixing times or by examining buildup curves over a range of mixing timesOther nOe caveats•I mentioned that nOe buildup rates are faster for larger proteins because of the longer correlation time•It’s also true that buildup rates can differ for nuclei within the same protein if different parts of the protein have different mobility (hence different correlation times)•for parts of the protein which are relatively rigid (such as the hydrophobic core) correlation times will more or less reflect that of the whole protein molecule--nOe buildup will be fast•disordered regions (at the N- or C-termini, for instance) may have much shorter effective correlation times and much slower nOe buildup as a consequence•the bottom line is, the actual nOe observed between two nuclei at a given distance r is often less than that expected on the basis of the overall molecular correlation time.The goal: translating NOESY crosspeak intensities into nOe distance restraints•because the nOe is not always a faithful reflection of the internuclear distance, one does not, in general, precisely translate intensities into distances!•instead, one usually creates three or four restraint classes which match a range of crosspeak intensities to a range of possible distances, e.g.class restraint description *for protein w/Mr<20 kDastrong 1.8-2.7 Å strong intensity in short tm (~50 ms*) NOESYmedium 1.8-3.3 Å weak intensity in short tm (~50 ms*) NOESYweak 1.8-5.0 Å only visible in longer mixing time NOESY•notice that the lower bound of 1.8 Å (approximately van der Waals contact) is the same in all restraint classes. This is because, for reasons stated earlier, atoms that are very close can nonetheless have very weak nOe’s, or even no visible crosspeak at all.Calibration of nOe’s•the crosspeak intensities are often calibrated against the crosspeak intensity of some internal standard where the internuclear distance is known. The idea of this is to figure out what the maximal nOe observable will be for a given distance. • ideally, one choosesan internal standardwhere the maximal nOewill be observed (i.e. something not undergoing a lot of motion)• this calibration can then be usedto set intensity cutoffs for restraint classes, often using a 1/r6 dependencetyrosine distancealways the same!Coupling constants and dihedral anglesThere exist relationships between three-bond scalar coupling constants 3J and the corresponding dihedral angles , called Karplus relations. These have the general form 3J = Acos2 + Bcos + Cfromp. 30EvanstextbookEmpirical Karplus relations in proteins • comparison of 3J values measured in solution with dihedral angles observed in crystal structures of the same protein allows one to derive empirical Karplus relations that give a good fit between the coupling constants and the anglescoupling constantsin solution vs. angles from crystal structure for BPTIthese twoquantitiesdiffer by 60°because they are defined differentlyfrom p. 167 Wuthrich textbookEmpirical Karplus relations in proteins•Here are some empirical Karplus relations:3JH,HN()= 6.4 cos2(- 60°) -1.4 cos(- 60°) + 1.93JH,H2()= 9.5 cos2(- 120°) -1.6 cos(- 120°) + 1.83JH,H3()= 9.5 cos2() -1.6 cos() + 1.83JN,H3()= -4.5 cos2(+ 120°) +1.2 cos(+ 120°) + 0.13JN,H2()= 4.5 cos2(- 120°) +1.2 cos(- 120°) + 0.1•Notice that use of the relations involving the hydrogens would require that they be stereospecifically assigned (in cases where there are two 