Magnetic Resonance Lectures for Chem 341 James Aramini, PhD. CABM 014A

Magnetic Resonance Lectures for Chem 341 James Aramini, PhD. CABM 014A jma@cabm.rutgers.edu " J.A. 12/11/13

Dec. 4 Dec. 9 Dec. 11" " Outline" " 1. Introduction / Spectroscopy Overview 2. NMR Spectroscopy Theory and Practice 3. Protein NMR & Resonance Assignment 4. NMR Structure Determination & " "Biological NMR Applications "

" Lecture #3 Overview" " 1. Protein NMR Structure Determination i. NMR data -> Restraints ii. Structure Calculation & Validation 2. Some Biological NMR Applications i. Residual Dipolar Couplings ii. NMR of large proteins iii. Relaxation and Dynamics

Protein NMR Structure Determination" OVERVIEW "" 13 C, 15 N-protein 2D, 3D, 4D NMR Spectral Analysis / Assignment Structure Calculation / Refinement

Protein NMR Structure Determination" THE LEVELS OF PROTEIN STRUCTURE "" 1 o" 2 o" 3 o" 4 o"

Protein NMR Structure Determination" Experimental NMR Constraints" "NMR ensemble " Distance - NOE - H-bonds Dihedral Angle - 13 C δ - J-couplings Orientational - RDCs Other - PRE/PCS Structure" Program" CYANA XPLOR UNIO ARIA CNS AMBER Rosetta

NMR Observable: NOE (d) - a through space correlation (<5 Å) distance restraint Coupling Constant (J) - through bond correlation dihedral angle restraint 1 H- 1 H NOE 4.1Å 2.9Å 3 J HNHα NH Hα Chemical Shift (δ) - very sensitive to local changes in environment dihedral angle restraint Residual Dipolar Coupling Constant (D) - X-Y bond vector orientation in weak alignment medium orientational restraint Hα D XY NH

I. Distance Restraints: 1 H- 1 H NOEs" Nuclear Overhauser Effect: change is intensity of 1 NMR signal is " another is irradiated" NOE depends on motion" Through-space dipole-dipole interaction" η" η i = (I-I o )/I o { 1 H}- 15 N NOE 2D noe 1 6 r ab 3Dʼs Estrada et al (2011)

I. Distance Restraints: 1 H- 1 H NOEs" through-space < 5Å" noe " 1 r ab 6 H N d αn O N A A 2 d αα Hα H N d NN O N d Nβ, d Nγ, A A 1 Hα H O A A 3 Hα H d αβ, d αγ, d αα i+4 d αβ(i, i+3) d αn(i, i+3) d NN(i, i+3) d αn (i, i+4) i-1 i i+3 i+2 i+1 C C N N d α(i)n(j) N N C C

I. Distance Restraints: 1 H- 1 H NOEs" Nomenclature Distances & 2 0 structure K. Wüthrich (1986) NOE patterns & 2 0 structure <5Å weak <4Å medium <2.5Å strong 3 J HNHα

I. Distance Restraints: 1 H- 1 H NOEs" 1 H- 1 H Distances & 2 0 structure i. α-helix H-bond " Diagnostics: 3D 15 N-NOESY 3D HNHA" - big sequential H N -H N NOEs" small 3 J HNHα " - H α -H N (i,i+2), (i,i+3), (i,i+4) " d NN" α-helix

I. Distance Restraints: 1 H- 1 H NOEs" 1 H- 1 H Distances & 2 0 structure ii. β-strands β-strands antiparallel parallel Diagnostics: 3D 15 N-NOESY 3D 13 C-NOESY 3D HNHA" - big sequential H α -H N NOEs" big cross-strand large 3 J HNHα " " " " " " H α -H α NOEs"

I. Distance Restraints: 1 H- 1 H NOEs" 1 H- 1 H Distances & 2 0 structure iii. β-turns β-turns Diagnostics: 3D 15 N-NOESY 3D HNHA" - specific sequential " " " specific 3 J HNHα " H N -H N and H α -H N NOEs " " pattern"

II. Dihedral Angle Restraints:" Protein chemical shifts depend on fold" " " Unfolded " " " " "" " " random " coil " " " " "" " " δ s Folded " " " " " "Local Environment" " " " " " "Secondary Structure"

II. Dihedral Angle Restraints:" 3 J HNHα & backbone dihedral angles" " " " " H " " " " "" α " 3 " " " " "" J HNHα " " " " " "" H N" 3D HNHA" β- ~8-10 Hz " J(φ-60) = 6.51cos 2 (φ-60) + -1.76cos(φ-60) + 1.60 α ~3-4 Hz " Vuister & Bax (1993) J. Am. Chem. Soc. 115, 7772

II. Dihedral Angle Restraints:" Chemical shifts & backbone dihedral angles" IFNγ " Cα and Cβ CSI " " " " " +1, 0, -1 vs. random coil δ ± range α-helix: ΔCα ~ +3 ppm ΔCβ ~ -1 ppm β-strand: ΔCα ~ -2 ppm ΔCβ ~ +3 ppm Spera & Bax (1991) J. Am. Chem. Soc. 113, 5490 Wishart & Sykes (1994) J. Biomol. NMR 4, 171

II. Dihedral Angle Restraints:" TALOS+: Φ,ψ restraints from δʼs "" Φ,ψ space using δʼs, residue type, neighbors " L8" INPUT: 13 C α, 13 C β, 13 C, 1 H α, 1 H N, 15 N "" OUTPUT: Φ,ψ; +/-; S 2 " Ex. Ubq" S 2 " Shen et al (2009) J. Biomol. NMR 44, 213 CSI "

Structure Determination:" Manual" Automated: CYANA, AutoStructure, ARIA, UNIO,." "Input Files: assignments (δʼs), NOE spectral peak lists" " " TALOS+ Φ/ψ, stereospecific assignments" " " (Other: H-bonds, Jʼs, RDCs, etc.)" " " " " " "" FGF-2 Montelione et al (2000) Nat Struct Mol Biol 7, 982

Iterative NMR Structure Refinement:" " " " " " "" Convergence" Constraints Convergence"

Structure Quality & Validation: Many Programs & Metrics " "" NMR Assignment & Restraint Stats Restraint Violations RMSD stats Rama- Global NOE chandran Quality Agreement

III. Residual Dipolar Couplings:" Global structural restraints" Weak alignment" "" RDC (D in Hz)" Dipole-Dipole Interaction"

III. Residual Dipolar Couplings:" Many alignment methods" i. Bicelles" " ii. Filamentous phage" iii. PAGE" Measure " ΔJ free aligned J NH J NH + D NH

III. Residual Dipolar Couplings:" RDC Theory" " 1 H" 15 N"

III. Residual Dipolar Couplings: Applications" Global Structural Information " " Structure Refinement" Helical Curvature Angle between 2 o Structural Elements IgG-binding domain of protein A no RDC" + RDC" Relative Orientation of Domains no RDC" + RDC" Zheng et al (2004) Protein Sci. 13, 549

NMR of Large Proteins / Complexes" Techniques for raising the MW Limit in Biomolecular NMR" TROSY TRansverse Optimized Deuteration/Selective Protonation SpectroscopY [ 2 H, 13 C, 15 N, 1 H-Ile-δ1,Leu-δ,Val-γ] Pervushin et al (1997) PNAS 94, 12366 select narrow " sub-peak" Goto et al (1999) J Biol NMR 13, 369-374. CAP Tzeng & Kalodimos (2012) Nature 488, 236

NMR Relaxation and Dynamics" Insights into a broad range of molecular motions" Boehr et al. (2006)

NMR Relaxation Analysis of Proteins" T 1, T 2, CPMG relaxation dispersion experiments T 1 inversion recovery T 2 CPMG CPMG relaxation dispersion I(τ) = I(0) (1 2e -τ/t 1) [ ] n I(T) = I(0) e -T/T 2 Baldwin & Kay (2009)

NMR Relaxation Analysis of Proteins" ns motion " " "ns - μs - ms motion" " Backbone Dynamics Conformational Entropy & Molecular Recognition Oligomerization State 13 CH 3 Relaxation Aramini et al (2010 & 2011) Tzeng & Kalodimos (2012) Nature 488, 236

THANK YOU" &" GOOD LUCK"