Biomolecular Nuclear Magnetic Resonance Spectroscopy BASIC CONCEPTS OF NMR How does NMR work? Resonance assignment Structural parameters 02/03/10 Reading: Chapter 22 in Protein and Peptide Drug Analysis “Solution Structure Determination of Proteins by NMR”

Nuclear Spin Nuclear spin angular momentum is a quantized property of the nucleus in each atom The nuclear spin angular momentum of each atom is represented by a nuclear spin quantum number (I) All nuclei with even mass numbers have I=0,1,2… All nuclei with odd mass numbers have I=1/2,3/2... NMR is possible with all nuclei except I=0 (e.g. 12 C), but I=1/2 has simplest spin physics Biomolecular NMR  primarily 1 H, 13 C, 15 N ( 31 P)

Spin 1/2 Nuclei in a Magnetic Field BoBo Energy  E = h  B o Nuclear efficiency factor: (gyro-magnetic ratio) ConstantsStrength of magnet

Intrinsic Sensitivity of Nuclei Nucleus  Natural Relative Abundance Sensitivity 1 H2.7 x C6.7 x N -2.7 x P1.1 x Prepare samples enriched in these nuclei

Variables Affecting Sensitivity -  E is very small   N is small -  N ~ 1:10 5 (at room T) NMR has low sensitivity  requires lots of sample!  E = h  H o Efficiency factor- nucleus ConstantsStrength of magnet NNNN = e -  E/kT Sensitivity (S) ~  pop. (N  vs N  ) S ~  N = Increase sensitivity by increasing magnetic field strength or reducing electronic noise (cryo-probes)

The Resonance Experiment BoBo Equilibrium EE h  E B1B1 Pump in energy (RF transmitter) Non-equilibrium Equilibrium h  E Release energy (RF receiver) NMR signals Strength of signal   (population)

NMR Terminology Chemical Shift & Linewidth The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus

Scalar and Dipolar Coupling  Coupling of nuclei gives information on structure Through Bonds Through Space

Resonance Assignment CH 3 -CH 2 -OH Which signal from which H atoms? OHCH 2 CH 3 Approach: use the scalar and dipolar couplings to match the set of signals with the molecular structure

2D NMR Spectroscopy Facilitates Identification of Coupling Partners Coupled spins F1F1 F2F2

Biomolecules Have Many Signals 1 H NMR Spectrum of Ubiquitin ~75 residues, ~500 1 H resonances  Terminology: signals are overlapped

Challenges For Using NMR to Study Biological Macromolecules Hundreds-thousands of signals! Must assign the specific signal for each atom Thousands of couplings between nuclei- these also need to be assigned

Critical Features of Protein NMR Spectra Regions of the spectrum correspond to different parts of the amino acid Tertiary structure leads to increased dispersion of resonances

Regions of the 1 H NMR Spectrum and Dispersion by the 3D Fold What would the unfolded protein look like?

Critical Features of NMR Spectra of Biomolecules Regions of the spectrum correspond to different parts of the amino acid Tertiary structure leads to increased dispersion of resonances Bio-macromolecules are polymers  The nuclei are coupled to some (but not all!) other nuclei

Spectra of Biomacromolecules: Overlapped Sub-Spectra *Groups of coupled nuclei* Each residue in the sequence gives rise to an independent NMR sub-spectrum, which makes the problems much simpler than if all spins were coupled to all other spins Methods have been developed to extract each sub-spectrum from the whole

Basic Strategy to Assign Resonances in a Protein 1.Identify resonances for each residue (scalar) 2.Put residues in order (dipolar, scalar) R - G - S - T - L - G - S LT G S S R G Same idea for any biopolymer (e.g. DNA, RNA)

Even Sub-Spectra are Overlapped!  Resolve resonances by multi-dimensional experiments 1 H NMR Spectrum of Ubiquitin

Solutions to the Overlap of Sub-spectra 1.Increase dimensionality of spectra to better resolve signals: 1D  2D  3D  4D….

Use of 2D NMR to Resolve Overlapping Sub-spectra in 1D 1D Sub-spectra overlapped 2D Coupled spins Off-diagonal cross peaks resolved!

Overlap in 2D NMR Spectra If 2D cross peaks overlap  go to 3D or 4D or …..

Solutions to the Challenge of Overlap in nD NMR Spectra 1.Increase dimensionality of spectra to better resolve signals: 1D  2D  3D  4D…. 2.Use hetero nuclei ( 13 C, 15 N) to distinguish 1 H cross peaks t2t2 t1t1 t3t3 HAHA HzHz

2D Heteronuclear NMR Spectroscopy 15 N- 1 H HSQC F1 Chemical Shift ( 15 N) F2 Chemical Shift ( 1 H) - 15 N - C  - CO H R

Advantages of Heteronuclear nD NMR Uses a second nucleus to resolve overlap of the first: chemical shift of each nucleus is sensitive to different factors More information to identify resonances Less sensitive to MW because this strategy uses large 1 and 2-bond scalar couplings