1 H NMR Spectra of Proteins

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1 NMR of Proteins

2 Determining Protein Structures by NMR the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands) of short protonproton distances and angles, and restraining the protein structure with these computationally H H.} d

3 1 H NMR Spectra of Proteins 1D, 1 H NMR spectra of even small proteins are impossible to interpret in any comprehensive manner -normally, only gross statements about secondary structure, tertiary structure, etc. can be made simple 1D 1 H experiment ubiquitin (76 amino acids, 8.5 kda) 2D 1 H COSY experiment cytochrome c, 12.5 kda COSY t 1 t 2 for even moderate sized proteins, addition of a second dimension still does not alleviate spectral crowding and overlap in 1 H spectra

4 nd, heteronuclear NMR Spectra of Proteins Modern NMR spectroscopic studies of proteins rely on multidimensional experiments involving 1 H, 13 C, and 15 N nuclei in isotopically labeled proteins These methods provide for signal selection (selectivity) and a means to reduce signal overlap simple 2D 1 H, 15 N HSQC experiment ubiquitin (76 amino acids, 8.5 kda) In order to measure the distances between protons, we need to find out what protons give rise to the signals in the spectra, I.e. we have to assign the protein (figure out the chemical shifts for all of the protons) The methods used are based on heteronuclear spectra

5 Triple Resonance Approach applicable to uniformly isotopically enriched proteins -uniform 13 C and 15 N labeling: spin 1/2 -three nuclei ( 1 H, 15 N, and 13 C) are involved based on magnetization transfer via (mostly) one bond J couplings -most of these couplings are large compared to linewidths for moderate sized proteins (~20 kda) -magnetization transfer is efficient -indirect ( 1 H) detection provides selective chemical shift correlation -spectral degeneracy minimized

6 Uniform Isotopic Labeling of Proteins Proteins can be uniformly isotopically labeled by recombinant expression using defined media -bacterial expression most common -also yeast, and cell-free systems are being developed -minimal media using 13 C 6 glucose as the sole carbon source and 15 NH 4 Cl (or -SO 4 ) as the sole nitrogen source -normally >98% atom excess -also labeled rich media ($$) -for larger proteins, uniform or fractional 2 H labeling also used - 2 H, 13 C glucose and D 2 O

7 1 J and 2 J Couplings in Proteins - these 1 J and 2 J couplings are uniform throughout polypeptides/proteins - these 1 J and 2 J couplings are virtually conformation independent

8 Prototypical Triple Resonance Experiment: HNCA correlates the chemical shifts of 1 H N, 15 N, 13 C α i and 13 C α i-1

9 Prototypical Triple Resonance Experiment: HNCA both 13 C α i and 13 C α i-1 chemical shifts are correlated -the peak for the intra-residue correlation is usually more intense (11 Hz 1 J NCα coupling vs 7 Hz 2 J NCα coupling) 1 H, 15 N-HSQC HNCA

10 Triple Resonance Approach: A Simple Example 2D HNCA projection 3D HNCA

11 Triple Resonance Approach: A Simple Example

12 Triple Resonance Approach: A Simple Example link the correlated shifts numerically.. or visually

13 Triple Resonance Approach: HNCA/HN(CO)CA Example problems: 13 C α chemical shift degeneracy in proteins 13 C α linewidths/resolution -these preclude complete linkage via 13 C α alone -the same is true for 13 C β, 13 C

14 The Nuclear Overhauser Effect The Nuclear Overhauser Effect or Nuclear Overhauser Enhancement is the change (enhancement) of the signal intensity from a given nucleus as a result of exciting or saturating the resonance frequency of another nucleus It is based on through-space interactions The magnitude of the effect is dependent on distance -enhancement depends on 1/r 6, where r is the internuclear distance -thus, the effect is limited to distances of approximately 5Å or less This provides a means to determine if any two protons in a protein are < 5Å apart The basic experiment used for proteins is called a NOESY cytochrome c, 12.5 kda 2D NOESY t 1 τ m t 2 Even for relatively small proteins, the 2D NOESY spectrum is hampered by severe spectral overlap

15 3D NOE Experiments for Distance Restraints -pulse sequences: combine 2D sequences to get 3D sequences -get increased dimensionality and increased resolution without an increase in the number of signals (peaks) NOESY t 1 τ m t 2 HSQC 1 H 15 N τ τ τ τ t 1 /2 t 1 /2 t 2 decouple 1 H t 1 τ m t 3 τ τ τ τ NOESY-HSQC 15 N t 2 /2 t 2 / decouple

16 3D NOE Experiments for Distance Restraints Left: 2D NOESY Far left: 2D plane of 3D NOESY- HMQC ( 1 H, 13 C) - 1 H signals resolved by 13 C chemical shifts of bound 13 C atoms

17 Structure Calculations the primary structural restraint information for high resolution protein structures are the NOE-based distance restraints -additional restraints include angle restraints based on coupling constants, long range restraints based on dipolar couplings, hydrogen bond restraints calculation of structures involves satisfying structural restraints using simulated annealing/ restrained molecular dynamics -a target/energy function including terms for covalent geometry (known bond lengths and bond angles) and experimental restraints is minimized -the molecule is computationally heated/cooled to attempt to find a global minimum the number of restraints is an important indicator of the quality of final structures

18 Practical Aspects of Protein Structure Determination using NMR

19 Introduction NMR vs X-ray crystallography for protein structure determination in an x-ray diffraction pattern, each datum (reflection) contains information about each atom in the asymmetric unit -each atom contributes information that contributes to the intensity of each reflection in an NMR spectrum, each datum (peak) contains information about only a single interatomic distance or angle -the process of determining a solution structure by NMR is one of measuring many small distances and angles one at a time Why use NMR? can t get a crystal / want to work in solution want to look at binding to other proteins/molecules want to understand stability want to measure fast dynamics processes

20 Introduction Some things you should know before you talk to an NMR spectroscopist about determining a structure Protein production Protein purity Isotopic labeling NMR samples / conditions / tubes Simple spectra / evaluation (stability, tertiary structure) Protein size / magnet size

21 Protein production protein sample(s) in theory, as little as a few mg of protein is sufficient -best if the protein sample for NMR is > 1 mm in practice, tens of milligrams (or more) are usually necessary, as are multiple samples -multiple samples if your protein is not stable -multiple samples with different isotopic labeling schemes many very good bacterial expression vectors/cell strains are available for expression in bacteria -good track record, easily automated expression in eukaryotic cells more complicated (yeast, insect, human cells) are cell-free systems currently available (i.e. Roche Rapid Translation System (RTS)).good for specific isotope labeling, not good for uniform isotopic labeling

22 Protein purity protein samples should always be as pure as possible in practice, for small proteins, small amounts of high molecular weight contaminants are OK SDS-PAGE molecular weight markers sample

23 Isotopic labeling of protein for NMR protein must be uniformly isotopically labeled with 13 C and 15 N not difficult these days: bacterial expression cell strains grow well on minimal media (D-glucose (U- 13 C 6, 98-99%) and 15 NH 4 Cl (98-99%) as sole carbon and nitrogen sources, respectively) for 1L medium, $300 for glucose (2 $150/g), and $30 for 15 NH 4 Cl (1 $30/g) there are also alternatives to minimal media (i.e. isotopically labeled rich media), but they are much more expensive

24 Isotopic labeling of protein for NMR in practice, often multiple samples with other isotopic labeling schemes are necessary 15 N only for certain angle (φ, ψ) measurements -also, usually used for initial evaluation of sample/spectra 13 C, 15 N plus partial or uniform deuteration for large proteins -requires growth in minimal media in D 2 O -cells must be adapted to growth on D 2 O samples with only specific amino acid types labeled assist in NMR resonance assignment -cells grown on medium with all unlabeled amino acids except for the one of interest -more common for larger proteins samples made with a mixture of 10% U- 13 C 6 glucose and 90% unlabeled glucose are used for stereospecific -CH 3 assignment -pror methyl groups of Valine and Leucine

25 Isotopic labeling of protein for NMR isotopic labeling to identify specific amino acid types, groups, or to assign stereospecificity 15 N-Gly only labeled protein uniformly 15 N labeled protein ε 13 CΗ 3 -Met only labeled protein uniformly 13 C labeled protein Left: sample prepared by growth on 100% uniformly 13 C-labeled glucose Right: sample prepared by growth on 10% uniformly 13 C-labeled glucose and 90% unlabeled glucose

26 The NMR sample buffer: no C- or N-bound protons phosphate is the best. deuterated Tris, deuterated acetate, deuterated imidazole, etc., are OK (can be expensive) salts K +, Na +, Cl -, SO 4 2-, etc., all OK (no protons) too much salt leads to decreased S/N ph: neutral or lower is best must minimize the rate of exchange of amide protons with solvent solvent 90% H 2 O, 10% D 2 O (for instrumental lock)

27 The NMR sample temperature sample dependent: usually 25 to 35 C bacteriostatic agents sodium azide used widely put it all together in a good quality, clean NMR tube standard NMR tube is 5 mm diameter (for use in a 5 mm NMR probe).volume of sample is ~ ul (~1 mm protein) magnetic susceptibility matched tubes, Shigemi tubes, permit lower volume samples to be used (i.e. less sample, or more concentrated sample), usually without deleterious effects (but the tubes are complicated) volume of sample is ul

28 Initial NMR spectra / evaluation 1D 1 H NMR spectrum of a small organic compound F(ω) = x x f (t)e iωt dt f (t) = 1 2π x x F(ω)e iωt dω 1D 1 H NMR spectrum of a small protein for even small proteins, 1D spectra are complicated and cannot be analyzed comprehensively 1D spectra can be useful, however, for evaluating the suitability/stability of a protein sample ubiquitin (76 amino acids, 8.5 kda)

29 Initial NMR spectra / evaluation 1D 1 H NMR spectrum of a small protein for properly folded small proteins -peaks should be sharp -peaks should show good chemical shift dispersion (i.e. tertiary structure intact) for unfolded proteins -peaks are usually broad (many protons in each peak) -chemical shift dispersion poor (leading to the broad peaks) acid-unfolded ubiquitin ubiquitin, neutral ph

30 Initial NMR spectra / evaluation sample stability can takes weeks of instrument time to acquire all data for structure determination sample has to be stable for the amount of time necessary to acquire all of the data (at the data acquisition temperature), plus the time between experiments (all data is rarely acquired all at once) protein x, t = 0 properly folded protein x, t = 2 weeks (at room temperature) partially unfolded

31 Initial NMR spectra / evaluation simple 2D 1 H, 15 N correlation NMR spectra of proteins reduce complex spectra to simple ones based on isotope editing reduce/eliminate spectral overlap/spectral degeneracy correlate amide 1 H- 15 N pairs ubiquitin (76 amino acids, 8.5 kda) simple 2D 1 H, 15 N HSQC experiment this spectrum demonstrates 1). that you can express your protein, 2). That you can isotopically label your protein, 3). That your protein is pure (1 peak per amino acid), 4) that your protein is folded (tertiary structure / good chemical shift dispersion), 5). etc. granting agencies need to see this spectrum or similar (akin to a crystal and a diffraction pattern)

32 Initial NMR spectra / evaluation tertiary structure and sample stability chemical shift dispersion and peakwidths reflect tertiary structure c: apomyoglobin b and a: acid unfolded apomyoglobin protein x, t = 0 properly folded protein x, t = 2 weeks (at room temperature) partially unfolded

33 Size (of the protein) matters the rotational correlation time (τ c ) scales with protein size larger τ c : peak broadening and decreased S/N larger proteins have more atoms, therefore more peaks in the spectra more peaks: increased peak overlap/chemical shift degeneracy ubiquitin (76 amino acids, 8.5 kda) AlgH (189 amino acids, 20.2 kda) EPSP synthase (427 amino acids, 46.2 kda)

34 Size (of the magnet) matters 1 H, 15 N-HSQC (TROSY) spectra of EPSP synthase (46 kda) at 600 and 800 MHz higher field means higher sensitivity (increased S/N), increased resolution (decreased peak overlap), and a bonus increase in S/N in TROSY experiments 600 MHz 800 MHz

35 ligand binding NMR: beyond structure slow dynamics / local and global stability (hydrogen exchange) residue mutational affects fast dynamics (ps/ns) via nuclear relaxation S 2 residue

36 END