NMR Spectral Assignment and Structural Calculations Lucia Banci CERM – University of Florence EMBO Global Exchange Course Suwon, Korea 19-26 June, 2016
Structure determination through NMR Protein Sample NMR spectroscopy Sequential resonance assignment Collection of conformational constraints 3D structure calculations Structure refinement and Analysis
The protein in the NMR tube! • Protein overexpression • Purification • 15 N/ 13 C labelling < 25 KDa 13 C, 15 N labeling about 240 AA 13 C, 15 N labeling > 25 kDa + 2 H labeling necessary!! about 240 AA Which experiments should I run to assess sample quality?
Is my sample OK for NMR? 1 H- 15 N HSQC spectra give the protein fingerprint folded unfolded 15 N 15 N NH 2 groups of ASN, GLN sidechains 1 H 1 H Folded proteins have larger dispersion Signals of unfolded proteins have little 1 H dispersion, that Can I see all the peaks I expect? means the 1 H frequencies of all residues are very similar. Count the peaks! Backbone NH (excluding prolines!)
Making resonance assignment What does it mean to make sequence specific resonance assignment ? HN(Asp2) HN i HN j HN(Leu50) N i N(Asp2) N j N(Leu50) C a i , C b i C a , C b (Asp2)..etc H a i , H b i H a , H b (Asp2) H a j , H b j H a , H b (Leu50) C a j , C b j , C g j ..etc C a , C b , C g 1 (Leu50)..etc To associate each resonance frequency to each atom of the individual residues of the protein
Assignment Strategy The strategy for assignment is based on scalar couplings
Experiments for backbone assignment 1 H i - 15 N i - 13 C a 13 C b i-1 i-1 CBCA(CO)NH and Res i-1 Res i CBCANH correlate amide groups (H and 15 N) with C a and C b resonances. 1 H i - 15 N i - 13 C a 13 C b i i 1 H i - 15 N i - 13 C a 13 C b i-1 i-1 Res i-1 Res i 1 H (i) - 15 N (i) - 13 C a (i) 1 H (i) - 15 N (i) - 13 CO (i-1) HNCO HNCA { 1 H (i) - 15 N (i) - 13 C a (i-1) 1 H (i) - 15 N (i) - 13 CO (i-1) HN(CA)CO { 1 H (i) - 15 N (i) - 13 C a (i-1) HN(CO)CA 1 H (i) - 15 N (i) - 13 CO (i)
Experiments for backbone assignment CBCA(CO)NH CBCANH The chemical shifts of C a and C b atoms can be used for a preliminary identification of the amino acid type.
Sequential Assignment The 'domino pattern' is used for the sequential assignment with triple resonance spectra CB CANH CBCA(CO)NH Green boxes indicate sequential connectivities from each amino acid to the preceeding one
Experiment for side-chain assignment 1 H i a , 1 H i b , 1 H i g 1 ……. Res i-1 Res i In H(C)CH-TOCSY, magnetization coherence is transferred, through 1 J couplings, from a proton to its carbon atom, to the neighboring carbon atoms and finally to their protons.
H(C)CH-TOCSY experiment F2 (ppm) 13 C F1 (ppm) 1 H C d C g 2 C g 1 C b C a Isoleucine 1 H F3 (ppm)
Conformational restraints NMR experimental data Structural restraints NOEs Proton-proton distances Coupling constants Torsion angles Torsion angles Chemical shifts H -bonds Proton-proton distances RDCs Bond orientations Relaxation times Metal-nucleus distances Metal-nucleus distances { PCSs Orientation in the metal frame Torsion angles Contact shifts
Distance constraints NOE is based on a relaxation process due to dipolar coupling between two nuclear spins. NOESY volumes are proportional to the inverse of the sixth power of the interproton distance (upon vector reorientational averaging)
The NOESY experiment: 1 H All 1 H within 5-6 Å from a 1 H can produce a cross-peak in NOESY spectra whose volume provides 1 H- 1 H distance restraints 15 N 1 H 1 H
How are the distance constraints obtained from NOEs intensities? CYANA NOEs calibration The NOESY cross-peak intensities (V) are converted into upper distance limits (r) through the relation: where K is a constant and n can vary from 4 to 6. K V = K constant is initially determined from NOE’s n r between protons at fixed distance log V = log K - n· log r .. .. . log V .. . . Classes of constraints . . . . . .... . . .. . 1. Backbone V = A/d 6 . ... 2. Sidechain V = B/d 4 3. Methyl V = C/d 4 Distances are given as value range log r Wuthrich, K. (1986) "NMR of Proteins and Nucleic Acids"
How are the distance constraints obtained from NOEs intensities? Xplor-NIH Calibration of NOEs The NOESY cross-peak intensities are converted into upper distance limits Classes of restraints Distance ranges 1. Very Weak 0 – 20% 1.8 – 6.0 Å 2. Weak 20 – 50% 1.8 – 5.0 Å 3. Medium 50 – 80% 1.8 – 3.3 Å 1.8 – 2.7 Å 4. Strong 80 – 100% 0.5 Å are added to the upper bound of distances involving methyl groups in order to correct for the larger than expected intensity of methyl crosspeaks J. J. Kuszewski, R. A. Thottungal, G. M. Clore, Charles D. Schwieters J Biol NMR 2008
Dihedral angles Backbone dihedral angles Sidechain dihedral angles
Dihedral angle restraints 3 J coupling constants are related to dihedral angles through the Karplus equation H a C a ψ H N 3 2 a = J ( HN H ) A cos ( 60 ) B cos( 60 ) C Karplus equation – 155 ° < < – 85 ° b strand conformation J HNH a > 8Hz – 70 ° < < – 30 ° a helix J HNH a < 4.5Hz 4.5Hz < J HNH a < 8Hz coil ,y are also determining the J HNC values
Chemical Shift Index As chemical shifts depend on the nucleus environment, they contain structural information. Correlations between chemical shifts of C a , C b ,CO, H a and secondary structures have been identified. Chemical Shift Index CSI’s are assigned as: C a and carbonil atoms chemical shift difference with respect to reference random coil values: -0.7 ppm < Dd < 0.7 ppm 0 Dd < - 0.7 ppm -1 Dd > +0.7 ppm +1 For C b the protocol is the same but with opposite sign than C a Any “dense” grouping of four or more “ -1 ’s”, uninterrupted by “ 1 ’s” is assigned as a helix, while any “dense” grouping of three or more “ 1 ’s”, uninterrupted by “ - 1 ’s”, is assigned as a b -strand. Other regions are assigned as “coil” . A “dense” grouping means at least 70% nonzero CSI’s .
H-bonds as Structural restraints HNCO direct method Experimental Determination of H-Bonds: H/D exchange indirect method Upper distance limit Distance and angle restraints Lower distance limit Distance between the donor and the a -Helix b Sheet acceptor atoms is in the range 2.7- 3.2 Å 140 ° < N-H···O < 180 °
Residual dipolar couplings Z B 0 Y X RDCs provide information on the orientation of (in principle each) bond-vector with respect to the molecular frame and its alignment in the magnetic field
Residual dipolar couplings D RDC f , IS i i i where is the molecular H alignment tensor with respect to the magnetic field and i , N are the angles between i the bond vector and the tensor axes Relative orientation of Proteins dissolved in liquid, orienting medium secondary structural Some media (e.g. bicelles, filamentous phage, elements can also be cellulose crystallites) induce to the solute some orientational order in a magnetic field determined A small “residual dipolar coupling” results
General Consideration How complete are the NMR structural restraints? NMR mainly determines short range structural restraints but provides a complete network over the entire molecule
3D structure calculations Most Common Algorithms • MD in cartesian coordinates/Simulated annealing X PLOR-NIH • MD in torsion angle space/Simulated annealing X PLOR-NIH and CYANA A random coil polypeptide chain is generated, which is folded through MD/SA calculations and applying experimental constraints
Molecular Dynamics (MD) How the algorithms work: • MD calculations numerically solve the equation of motion to obtain trajectories for the molecular system • In Cartesian coordinates, the Newton‘s equation of motion is: E hybrid = w i • E i • In torsion angle space the equations of motion (Lagrange equations) are = w bond •E bond + w angle •E angle + w dihedral • E dihedral + solved in a system with N torsion angles as the only degrees of freedom. w improper •E improper + w vdW •E vdW + Conformation of the molecule is uniquely specified by the values of all w NOE •E NOE + w torsion •E torsion + ... torsion angles. About 10 times less degrees of freedom than in Cartesian space L = E kin – E pot d L L = 0 q = generalized dt coordinate q q k k
How MD is used to find the lowest energy conformation? • The potential energy landscape of a protein is very complex and studded with many local minima where a conformation can become “trapped” during MD calculations E hybrid = w i • E i = w bond •E bond + w angle •E angle + w dihedral • E dihedral + • A distinctive feature of MD simulations, when w improper •E improper + w vdW •E vdW + compared to the straightforward minimization of an w NOE •E NOE + w torsion •E torsion + ... energy function, is the presence of kinetic energy that allows the protein conformations to cross barriers of the potential surface •
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