Скачать презентацию BCHM 313 Physical Biochemistry Dr Michael Nesheim Скачать презентацию BCHM 313 Physical Biochemistry Dr Michael Nesheim

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BCHM 313 – Physical Biochemistry Dr. Michael Nesheim (Coordinator) nesheimm@queensu. ca Rm. A 210 BCHM 313 – Physical Biochemistry Dr. Michael Nesheim (Coordinator) [email protected] ca Rm. A 210 Botterell Dr. Steven Smith (Co-coordinator) steven. [email protected] ca Rm. 615 Botterell Dr. Susan Yates [email protected] ca Rm. 623 Botterell

BCHM 313 – Physical Biochemistry Topics 1. Protein NMR – Smith 2. Macromolecular Crystallography BCHM 313 – Physical Biochemistry Topics 1. Protein NMR – Smith 2. Macromolecular Crystallography – Yates 3. Hydrodynamics – Nesheim 4. Equilibrium Binding – Nesheim 5. Enzyme Kinetics – Nesheim 6. Spectroscopy – Nesheim 7. Evaluation 8. Midterm test – 35% 9. Final exam – 65%

Protein NMR Spectroscopy Determining three-dimensional structures and monitoring molecular interactions (http: //pldserver 1. biochem. Protein NMR Spectroscopy Determining three-dimensional structures and monitoring molecular interactions (http: //pldserver 1. biochem. queensu. ca/~rlc/steve/313/)

Outline • N-dimensional NMR • Resonance assignment in proteins • NMR-based structure determination • Outline • N-dimensional NMR • Resonance assignment in proteins • NMR-based structure determination • Molecular interactions Reference textbooks: - Lehninger - others available in my office.

Nuclear Magnetic Resonance (NMR) • MRI – Magnetic Resonance Imaging (water) • In-vivo spectroscopy Nuclear Magnetic Resonance (NMR) • MRI – Magnetic Resonance Imaging (water) • In-vivo spectroscopy (metabolites) • Soild-state NMR (large structures) • Solution NMR • Chemical structure elucidation - Natural product chemistry - Synthetic organic chemistry – analytical tool of choice for chemists • Biomolecular structural studies (3 D structures) - Proteins - DNA and Protein/DNA complexes - Polysaccharides • Molecular interactions - Ligand binding and screening (Biotech and Bio. Pharma) • Nobel prizes (3): Felix Bloch, 1952 (Physics); Richard Ernest, 1991 (Chemistry); Kurt Wuthrich, 2002 (Chemistry)

Spectroscopy & Nuclear Spin • Absorption (or emission) spectroscopy (IR, UV, vis). Detects the Spectroscopy & Nuclear Spin • Absorption (or emission) spectroscopy (IR, UV, vis). Detects the absorption of radiofrequencies (electro-magnetic radiation) by certain nuclei in a molecule - NMR • Unfortunately, some quantum mechanics are needed to understand it • Only nuclei with spin number (I) 0 can absorb/emit electro- magnetic radiation • Nuclei with even mass number & even number of protons (12 C): I=0 • With even mass number & odd number of protons (14 N): I = 1, 2, 3…. • With odd mass number (1 H, 15 N, 13 C, 31 P): I = 1/2, 3/2 ……. • Spin states of nucleus (m) are quantified: m. I = (2 I + 1) Thus, for biologically relevant nuclei, two spin states exist: 1/2, 1/2

Spin ½ Nuclei Align in Magnetic Fields Bo Energy Efficiency factornucleus DE = h Spin ½ Nuclei Align in Magnetic Fields Bo Energy Efficiency factornucleus DE = h Bo/2 Constants Strength of magnet • In ground state all nuclear spins are disordered – no energy difference degenerate • Since nuclei are small magnets, they orient when a strong magnetic field is applied – small excess aligned with field (lower energy)

Intrinsic Sensitivity of Nuclei Nucleus % Natural Abundance Relative Sensitivity 1 H 2. 7 Intrinsic Sensitivity of Nuclei Nucleus % Natural Abundance Relative Sensitivity 1 H 2. 7 x 108 99. 98 13 C 6. 7 x 107 1. 11 0. 004 15 N -2. 7 x 107 0. 36 0. 0004 31 P 1. 1 x 108 100. 1. 0 0. 5

Resonance: Perturb Equilibrium β Bo DE 1. equilibrium Efficiency factornucleus α H 1 h Resonance: Perturb Equilibrium β Bo DE 1. equilibrium Efficiency factornucleus α H 1 h = DE DE = h Bo/2 2. pump in energy Constant β α 3. non-equilibrium Strength of magnet

Return to Equilibrium (Relax) β DE 3. Non-equilibrium α h = DE 4. release Return to Equilibrium (Relax) β DE 3. Non-equilibrium α h = DE 4. release energy (detect) β 5. equilibrium α

Magnetic Resonance Sensitivity (S) ~ (population) Nβ = e-DE/k. T S ~ DN = Magnetic Resonance Sensitivity (S) ~ (population) Nβ = e-DE/k. T S ~ DN = Nα • E is small - @ r. t. ~1: 105 Thus, intrinsically low sensitivity *Need lots of sample Efficiency factornucleus DE = h Bo/2 Constant Strength of magnet Increase sensitivity by increasing magnetic field strength

Energy/Frequency Relationship For a particular nucleus: DE = h B /2 = B /2 Energy/Frequency Relationship For a particular nucleus: DE = h B /2 = B /2 Have to consider precession since: 1) nucleus has inherent spin and 2) application of a large external magnet generates a torque which results in precession o Precession occurs around B at frequency, termed Larmor frequency ( ). m Bo = B /2 = Reason why 14. 1 Telsa magnet often called 600 MHz magnet.

Bulk Magnetization Two spins All spins Sum z x Bo b z z y Bulk Magnetization Two spins All spins Sum z x Bo b z z y Mo x z y x y y x

Power of Fourier Transform z z x 90 RF pulse y z t x Power of Fourier Transform z z x 90 RF pulse y z t x y = B B A t f NMR frequency Fourier Transform Variation of signal at X axis vs. time

Pulse Fourier Transform NMR z z x x y y z z 90 RF Pulse Fourier Transform NMR z z x x y y z z 90 RF pulse x t x y y 1 = B 2 = B A f 2 1 NMR frequency domain Spectrum of frequencies t Fourier Transform NMR time domain Variation in amplitude vs. time

Pulse FT NMR Experiment 90º pulse Experiment equilibration acquisition (t) equilibration Data Analysis FID Pulse FT NMR Experiment 90º pulse Experiment equilibration acquisition (t) equilibration Data Analysis FID Time domain (t) detection of signals Fourier Transform

NMR terminology • Magnetic field (B ) felt by each nucleus affected by its NMR terminology • Magnetic field (B ) felt by each nucleus affected by its local electronic environment - big difference between B (MHz) and Blocal (hundreds of Hz) ie. parts per million (ppm, ) • Use relative scale and refer all signals in spectrum to a signal from a reference compound (DSS) - ref = ref

Summary All spins Sum z Bo b } z y y = B = Summary All spins Sum z Bo b } z y y = B = z FID – time domain x t 90 RF x y DE = h z x Mo x y DE = h Bo/2 z x y

Summary (cont) FID – time domain frequency domain 10 Chemical shift – relative scale Summary (cont) FID – time domain frequency domain 10 Chemical shift – relative scale ppm 0

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

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

Proteins Have Too Many Signals! 1 H 1 D NMR Spectrum of Ubiquitin ~500 Proteins Have Too Many Signals! 1 H 1 D NMR Spectrum of Ubiquitin ~500 resonances 1 H (ppm) Resolve resonances by multi-dimensional experiments

Examples of Amino Acids Examples of Amino Acids

NMR experiment 90º pulse 1 D equilibration acquisition (t) equilibration 2 D 90º pulse NMR experiment 90º pulse 1 D equilibration acquisition (t) equilibration 2 D 90º pulse preparation Same as 1 D experiment detection of signals 2 D detect signals twice (before/after couple) evolution (t 1) mixing acquisition (t 2) Transfers between coupled spins

2 D NMR: Coupling is the Key 2 D detect signals twice (before/after couple) 2 D NMR: Coupling is the Key 2 D detect signals twice (before/after couple) 90º pulse evolution (t 1) preparation mixing acquisition (t 2) Transfers between coupled spins Same as 1 D experiment t 1 t 2

2 D NMR Spectrum excitation Pulse sequence preparation evolution (t 1) mixing acquisition (t 2 D NMR Spectrum excitation Pulse sequence preparation evolution (t 1) mixing acquisition (t 2) Spectrum HB Either: t 1 Before mixing HA Coupled spins or: t 1 After mixing t 2

The Power of 2 D NMR Resolving Overlapping Signals 1 D 2 signals overlapped The Power of 2 D NMR Resolving Overlapping Signals 1 D 2 signals overlapped 2 D 2 cross peaks resolved

Multi-Dimensional NMR Built on the 2 D Principle 3 D detects signals 3 times Multi-Dimensional NMR Built on the 2 D Principle 3 D detects signals 3 times 90º pulse excitation preparation evolution (t 1) Same as 1 D experiment mixing evolution (t 2) mixing t 1 acquisition (t 3) t 2 t 3

Protein NMR: Practical Issues Hardware: • Magnet: homogeneous, high field - $$$$ • Electronics: Protein NMR: Practical Issues Hardware: • Magnet: homogeneous, high field - $$$$ • Electronics: stable, tunable • Environment: temperature, pressure, humidity, stray fields Sample Preparation: • Recombinant protein expression (E. coli, Pichia pastoris etc) • Volume: 300 L – 600 L • Concentration: 1 D ~ 50 M, n. D ~ 1 m. M ie. @ 20 k. Da, 1 m. M = 10 mg • Purity: > 95%, buffers • Sensitivity ( ): isotope enrichment (15 N, 13 C)

Protein NMR: Practical Issues (cont. ) Solution Conditions: • Variables: buffer, ionic strength, p. Protein NMR: Practical Issues (cont. ) Solution Conditions: • Variables: buffer, ionic strength, p. H, temperature • Binding studies: co-factors, ligands • No crystals! Molecular Weight: • up to 30 – 40 k. Da for 3 D structure determination • > 100 k. Da: uniform deuteration, residue and site-specific, atomspecific labeling • Symmetry reduces complexity: 2 x 10 k. Da 20 k. Da

NMR Spectrum to 3 D structure? | 12 1 H (ppm) | 0 NMR Spectrum to 3 D structure? | 12 1 H (ppm) | 0

Critical Features of Protein NMR Spectra • The nuclei are not mutually coupled Each Critical Features of Protein NMR Spectra • The nuclei are not mutually coupled Each amino acid gives rise to an independent NMR sub-spectrum, which is much simpler than the complete protein spectrum • Regions of the spectrum correspond to different parts of the amino acid • Tertiary structure leads to increased dispersion of resonances • chemical shifts associated with each nucleus influenced by local chemical environment – nearby nuclei

Regions of a protein 1 H NMR Spectrum What would an unfolded protein look Regions of a protein 1 H NMR Spectrum What would an unfolded protein look like?

Solutions to the Challenges 1. Increase dimensionality of spectra to better resolve signals: 1 Solutions to the Challenges 1. Increase dimensionality of spectra to better resolve signals: 1 2 3 4 2. Detect signals from heteronuclei (13 C, 15 N) Ø Better resolution of signals/chemical shifts not correlated nuclei Ø More information to identify signals Ø Lower sensitivity to MW of protein

1 D Protein 1 H NMR Spectrum 1 D Protein 1 H NMR Spectrum

Resolve Peaks by Multi-D NMR A BONUS regions in 2 D spectra provide protein Resolve Peaks by Multi-D NMR A BONUS regions in 2 D spectra provide protein fingerprints If 2 D cross peaks overlap go to 3 D

Basic Strategy to Assign Resonances in Protein 1. Assign resonances for each amino acid Basic Strategy to Assign Resonances in Protein 1. Assign resonances for each amino acid T L G S S R G 2. Put amino acids in order - Sequential assignment (R-G-S, T-L-G-S) - Sequence-specific assignment 1 2 3 4 5 6 7 R-G-S-T-L-G-S

Acronyms for Basic Experiments Differ Only in the Nature of Mixing Homonuclear HSQC Heteronuclear Acronyms for Basic Experiments Differ Only in the Nature of Mixing Homonuclear HSQC Heteronuclear TOCSY (thru-bond) COSY COrrelation Spectroscop. Y Scalar Coupling Heteronuclear Hetero-TOCSY TOtal Correlation Spectroscop. Y Dipolar Coupling (thru-space) NOESY Nuclear Overhauser Effect (Enhancement) Spectroscop. Y NOESY-HSQC

Homonuclear 1 H Assignment Strategy • For proteins up to ~ 10 k. Da Homonuclear 1 H Assignment Strategy • For proteins up to ~ 10 k. Da • Scalar couplings to identify resonances/spin systems/amino acids, dipolar couplings to place in sequence • Based on backbone HN (unique region in 1 H spectrum, greatest dispersion of resonances, least overlap) • Concept: Build out from the backbone to identify the side-chain resonances (unique spin systems) • 2 nd dimension resolves overlap, 3 D rare

Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System COSY (3 -bond) TOCSY Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System COSY (3 -bond) TOCSY – – H – H C –H N–C–C = H H H O Alanine HN CH 3

Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System ’CH 3 COSY (3 Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System ’CH 3 COSY (3 -bond) CH 3 – – – H 3 C CH 3 TOCSY H C–H b’H b. H H–C–H N–C–C H = H H O Leucine HN

Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System CH 3 –H H Homonuclear 1 H Assignment Strategy Step 1: Identify Spin System CH 3 –H H O b’H H–C–H b. H –N–C–C = – N–C–C H CH 3 H H H H = – – C CH 3 TOCSY C–H H H – – – H 3 C ’CH 3 COSY (3 -bond) H O Alanine Leucine - open circles - closed circles HN HN

Homonuclear 1 H Assignment Strategy Step 2: Fit residues in sequence Minor Flaw: All Homonuclear 1 H Assignment Strategy Step 2: Fit residues in sequence Minor Flaw: All NOEs mixed together! Use only these to make sequential assignments Long Range Sequential Intraresidue • Sequential NOEs HN-HN (i, i + 1) H -HN (i, i + 1) A B C D • • Medium-range (helices: H -HN (i, i + 3, 4))) Z

Homonuclear 1 H Assignment Strategy Step 2: Fit residues in sequence ’CH 3 C–H Homonuclear 1 H Assignment Strategy Step 2: Fit residues in sequence ’CH 3 C–H C –H – H H H–C–H O H H CH 3 b. CH 3 H b’H b. H –N–C–C = N–C–C NOESY = COSY/TOCSY + H = – – H H – – – H 3 C H O HN HN

Extended Homonuclear 1 H Strategy • For proteins up to ~ 15 k. Da Extended Homonuclear 1 H Strategy • For proteins up to ~ 15 k. Da • Same basic idea as 1 H strategy: based on backbone HN • Concept: When backbone 1 H overlaps disperse with backbone 15 N • Use heteronuclear 3 D experiments to increase signal resolution 1 H 1 H 15 N

Solutions to the Challenges 1. Increase dimensionally of spectra to better resolve signals: 1 Solutions to the Challenges 1. Increase dimensionally of spectra to better resolve signals: 1 2 3 4 2. Detect signals from heteronuclei (13 C, 15 N) Ø Labeling with NMR-observable 13 C, 15 N isotopes Ø Better resolution of signals/chemical shifts not correlated nuclei Ø More information to identify signals Ø Lower sensitivity to MW of protein

Isotopic Labeling • Require uniform 15 N/13 C labeling ie. Every carbon and nitrogen Isotopic Labeling • Require uniform 15 N/13 C labeling ie. Every carbon and nitrogen isotopically labeled How? • Grow bacteria on minimal media (salts) supplemented with 15 N-NH Cl and 13 C-glucose as soles sources of nitrogen and 4 carbon • lower yields than protein expression than on enriched media, therefore need very good recombinant expression system

Double Resonance Experiments Increases Resolution/Information Content Double Resonance Experiments Increases Resolution/Information Content

Heteronuclear NMR: 15 N-Edited Experiments Increases Resolution/Information Content H R 15 N H – Heteronuclear NMR: 15 N-Edited Experiments Increases Resolution/Information Content H R 15 N H – C – 15 N – C O R

3 D Heteronuclear NMR: 15 N-Edited Experiments + 3 D Heteronuclear NMR: 15 N-Edited Experiments +

Extended Homonuclear 1 H Strategy 15 N dispersed 1 H-1 H TOCSY 3 overlapped Extended Homonuclear 1 H Strategy 15 N dispersed 1 H-1 H TOCSY 3 overlapped NH resonances (diagonal) m) 1 H p (p HN (ppm) Same NH, different 15 N F 3 F 2 F 1 TOCSY HSQC 1 H 1 H t 1 t 2 15 N t 3 H R 15 N H – C – 15 N – C O R

Summary of Homonuclear Assignment Strategy • for proteins up to ~10 k. Da (2 Summary of Homonuclear Assignment Strategy • for proteins up to ~10 k. Da (2 D homonuclear) and proteins up to ~ 15 k. Da (15 N-labeling and 3 D) • using scalar coupling-type experiments (COSY, TOCSY) assign spin systems/side-chain resonances • Connect amino acids (identified based on spin systems) sequentially using NOE-type experiments and characteristic sequential NOEs (HN-HN (i, i+1); H -HN (i, i+1))

Heteronuclear (1 H, 13 C, 15 N) Strategy • for larger proteins (backbone assignment: Heteronuclear (1 H, 13 C, 15 N) Strategy • for larger proteins (backbone assignment: ~70 k. Da; full structure determination: ~40 k. Da) • Assign resonances (chemical shifts) for all atoms (except O) 15 N • Handles overlap in backbone H region disperse with backbone C’, C , H , Cb, Hb • Heteronuclear 3 D/4 D increases resolution 1 H 13 C 1 H 15 N • Works on bigger proteins because scalar couplings are larger

Heteronuclear (1 H, 13 C, 15 N) Strategy Step 1: Sequence-specific backbone assignment Assign Heteronuclear (1 H, 13 C, 15 N) Strategy Step 1: Sequence-specific backbone assignment Assign backbone 1 H, 15 N, C , Cb resonances/chemical shifts and sequentially link amino acids using partner scalar coupling experiments Step 2: Side-chain assignment Assign side-chain 13 C & 1 H resonances/chemical shifts using TOCSY-type 3 D scalar coupling experiments ** Have complete list of chemical shifts for all 13 C, 15 N, 1 H atoms in protein **

Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments Names of scalar experiments Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments Names of scalar experiments based on atoms detected Consecutive residues!! NOESY not needed

Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments CBCA(CO)NH - inter-residue connectivity Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments CBCA(CO)NH - inter-residue connectivity (HN to previous C , Cb) HNCACB - intra-residue connectivity (HN to own C , Cb) Search 15 N planes for 13 C and 13 Cb chemical shifts 13 Cb R H H–C–H chemical shift H O H 13 C H O H H = H = N–C–C–N–C–C O chemical shift common 15 N and HN chemical shift in both experiments (found on same 15 N plane)

Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments CBCA(CO)NH - inter-residue connectivity Heteronuclear (1 H, 13 C, 15 N) Assignments Backbone Experiments CBCA(CO)NH - inter-residue connectivity (HN to previous C , Cb) HNCACB - intra-residue connectivity and possibly inter-residue (HN to own C , Cb) Start with unique residue 1. Gly – only C 2. Ala – upfield-shifted Cb (~18 ppm) 3. Thr/Ser – downfield-shifted C & Cb which are close to each other

Heteronuclear (1 H, 13 C, 15 N) Assignments Side-chain Experiments Multiple redundancies increase reliability Heteronuclear (1 H, 13 C, 15 N) Assignments Side-chain Experiments Multiple redundancies increase reliability

Heteronuclear (1 H, 13 C, 15 N) Assignments Key Points • Enables the study/assignment Heteronuclear (1 H, 13 C, 15 N) Assignments Key Points • Enables the study/assignment of much larger proteins (up to ~100 k. Da) • Scalar coupling-type 3 -dimensional experiments only • Bonus: Amino acid identification and sequence-specific assignment all at once • Most efficient but experiments are more complex • Requires 13 C, 15 N enrichment (also 2 H) High expression levels on minimal media Increased cost ($150/g 13 C-gluocose; $30/g 15 NH 4 Cl)

Structure Determination Overview List of chemical shifts for all nuclei in protein (1 H, Structure Determination Overview List of chemical shifts for all nuclei in protein (1 H, 13 C, 15 N)

NMR Experimental Observables Provide Structural Information 1. Backbone conformation from chemical shifts (Chemical Shift NMR Experimental Observables Provide Structural Information 1. Backbone conformation from chemical shifts (Chemical Shift Index – CSI; H , Cb, C’) 2. Hydrogen bond constraints 3. Backbone and side chain dihedral angle constraints from scalar couplings 4. Distant constraints from NOE connectivities

1. Chemical Shift Index • Comparison of H , Cb, C’ determined chemical shifts 1. Chemical Shift Index • Comparison of H , Cb, C’ determined chemical shifts from protein to standard random coil chemical shift values • Upfield-shifted H and Cb and downfield-shifted C and C’ values indicate amino acid residues in an -helical conformation (requires three consecutive residues displaying this pattern) • Downfield-shifted H and Cb and upfield-shifted C and C’ values indicate residues in an extended (b-strand) conformation

2. Hydrogen Bonds C=O • Slow rate of exchange of labile HN with solvent 2. Hydrogen Bonds C=O • Slow rate of exchange of labile HN with solvent • Protein dissolved in 2 H O; HN signals 2 disappear with time • HN groups that are Hbonded (i. e. part of secondary structure) will exchange a lot slower than those in loops H-N

3. Dihedral Angles from Scalar Couplings • • 6 Hz Ø Must accommodate multiple 3. Dihedral Angles from Scalar Couplings • • 6 Hz Ø Must accommodate multiple solutions multiple J values

4. 1 H-1 H Distances from NOEs Long-range (tertiary structure) Sequential Intraresidue A B 4. 1 H-1 H Distances from NOEs Long-range (tertiary structure) Sequential Intraresidue A B C D • • Z Medium-range (helices) Challenge is to assign all peaks in NOESY spectra - semi-automated processes for NOE assignment using NOESY data and table of chemical shifts yet still significant amount of human analysis

Protein Fold without Full Structure Calculations 1. Determine secondary structure • CSI directly from Protein Fold without Full Structure Calculations 1. Determine secondary structure • CSI directly from assignments • Medium-range NOEs 2. Add key long-range NOEs to fold

Approaches to Identifying NOEs • 1 H-1 H • 15 N- NOESY 2 D Approaches to Identifying NOEs • 1 H-1 H • 15 N- NOESY 2 D 1 H 1 H or 13 C dispersed 1 H-1 H NOESY 1 15 H 4 D 15 N C 1 H 1 H 13 C 1 H H 13 N 1 1 1 H H 3 D 13 C 1 H 1 H 13 C 15 N 1 H 15 N

NMR Structure Calculations Objective: Determine all conformations consistent with experimental data • Programs that NMR Structure Calculations Objective: Determine all conformations consistent with experimental data • Programs that only do conformational search may lead to bad geometry use simulations guided by experimental data • need a reasonable starting structure • Distance restraints arrived at from NOE signal intensities signal is an average of all conformations

NMR Structure Calculations (cont) 1. NOE signals are time & population-averaged (ie. measured on NMR Structure Calculations (cont) 1. NOE signals are time & population-averaged (ie. measured on entire sample over period of time) 2. Intensity of NOE signal 1 H-1 H distance (1/r 6) NOE distance restraints are given a range of values strong NOE: 0 - 2. 8 Å medium NOE: 2. 8 – 3. 5 Å weak NOE: 3. 5 – 5. 0 Å NMR data not perfect: Noise, incomplete data multiple solutions (conformational ensemble unlike X-ray crystallography with one solution)

Variable Resolution of Structures • Secondary structures well defined, loops variable • Interiors well Variable Resolution of Structures • Secondary structures well defined, loops variable • Interiors well defined, surfaces more variable • Trends the same for backbone and side chains Ø More dynamics at loops/surface Ø Constraints in all directions in the interior

Assessing the Quality of NMR Structures • Number of experimental constraints • RMSD of Assessing the Quality of NMR Structures • Number of experimental constraints • RMSD of structural ensemble (subjective!) • Violation of constraints- number, magnitude • Molecular energies • Comparison to known structures: PROCHECK • Back-calculation of experimental parameters

Summary of Protein NMR Structure Determination Sample preparation with possible isotope labeling Data collection Summary of Protein NMR Structure Determination Sample preparation with possible isotope labeling Data collection (scalar coupling and dipolar coupling expts. Resonance and sequence-specific assignments Identification and quantification of NOE peaks and intensities and conversion to approx. 1 H-1 H distances Generation of models consistent with NOE distance constraints, dihedral angle ranges, H-bond distances Model improvement by inclusion of newly identified NOES using above mentioned models

NMR Structures – Now what? NMR Structures – Now what?

Monitoring Molecular Interactions 15 N-1 H HSQC G 27 NMR Provides G 22 A Monitoring Molecular Interactions 15 N-1 H HSQC G 27 NMR Provides G 22 A 14 Ø Multiple probes Y 36 V 19 Ø Site-specific I 24 S 16 Ø In-depth info V 29 T 23 M 20 K 33 F 28 H 31 W 17 C 37 L 15 R 25 A 32 Q 21 I 34 D 18 N 30 S 35 K 26 Ø Spatial distribution of responses can be mapped on structure

Monitoring Molecular Interactions Titration followed by 15 N-1 H HSQC Monitoring Molecular Interactions Titration followed by 15 N-1 H HSQC

Monitoring Molecular Interactions Transcription factor (CBP) -oncoprotein (E 2 A) interaction - collaboration with Monitoring Molecular Interactions Transcription factor (CBP) -oncoprotein (E 2 A) interaction - collaboration with Dr. David Le. Brun (Pathology) Map of chemical shift perturbations on the structure of protein?

Monitoring Molecular Interactions - Identification of ligand (E 2 A)-binding site on the structure Monitoring Molecular Interactions - Identification of ligand (E 2 A)-binding site on the structure of the KIX domain of CBP

Monitoring Molecular Interactions Chemical Perturbation Mapping Structure Monitoring Molecular Interactions Chemical Perturbation Mapping Structure

Ligand Binding NMR timescale – 1 sec to 1 x 10 -6 sec 1/koff Ligand Binding NMR timescale – 1 sec to 1 x 10 -6 sec 1/koff = t >> 1 sec slow exchange, superposition of spectra 1/koff = t << 1 x 10 -6 sec fast exchange, weighted average A kon koff B Kdiss = [A]/[B] = koff/kon Ligand Binding - Another protein - Metal ion - Drug or chemical P + L = PL Kdiss = [P] [L] [PL]

Ligand Binding - exchange E 641, S 642, and S 670 - Fast exchange Ligand Binding - exchange E 641, S 642, and S 670 - Fast exchange (weighted average of free and bound populations) T 614 - Intermediate-fast exchange

Ligand Binding Ptot = P + PL Ltot = L + PL So……. Kdiss Ligand Binding Ptot = P + PL Ltot = L + PL So……. Kdiss = [Ptot - PL] [Ltot - PL] [PL] Plot [Ltot]/[Ptot] vs “change” in NMR spectra For fast exchange (weak binding): Change = obs - init sat - init = [ PL] [Ptot] shifting of resonances in spectra For slow exchange (tight binding): Change = Integral of peakobs Integral of peakmax = [ PL] [Ptot] intensity changes in peaks of free and bound forms

Monitoring Molecular Interactions Binding Constants by NMR Stronger Weaker Molar ratio of d-CTTCA Fit Monitoring Molecular Interactions Binding Constants by NMR Stronger Weaker Molar ratio of d-CTTCA Fit change in chemical shift to binding equation

Protein Dynamics Interesting because……. . • Function requires motion/kinetic energy • Entropic contributions to Protein Dynamics Interesting because……. . • Function requires motion/kinetic energy • Entropic contributions to binding events • Protein folding/unfolding • Uncertainty in NMR and crystal structures • Effects on NMR experiments: spin relaxation is dependent on motions know dynamics to predict outcomes and design new experiments

Characterizing Protein Dynamics Parameters & Timescale Characterizing Protein Dynamics Parameters & Timescale

Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states Populations ~ relative stability Rex < w (A) - w (B) Rate A B

Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW)

Linewidths Dependent on Protein MW A B A 15 N B 15 N 1 Linewidths Dependent on Protein MW A B A 15 N B 15 N 1 H 1 H 1 H • Same chemical shifts, • Linewidth determined same structure By size of molecule • Fragments have narrow linewidths

Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW) • Exchange of HN solvent: slow timescales (milliseconds to years!) • requires local or global unfolding events • HN involved in H-bonds exchanges slowly • surface or flexible region: HN exchange rapidly

Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow Dynamics from NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = fast motions, wide = slow motions; dependent on protein molecular weight (MW) • Exchange of HN solvent: slow timescales (milliseconds to years!) • NMR relaxation measurements (ps – ns; s – ms) • R 1 (1/T 1) spin lattice relaxation rate (z-axis) • R 2 (1/T 2) spin relaxation rate (xy-plane) • Heteronuclear NOE (15 N-1 H)

Dynamics to Probe the Origin of Structural Uncertainty Weak correlation Strong correlation - Measurements Dynamics to Probe the Origin of Structural Uncertainty Weak correlation Strong correlation - Measurements show if high RMSD is due to high flexibility (low S 2)

NMR and Crystallography NMR X-ray • Can mimic biological conditions - p. H, temp, NMR and Crystallography NMR X-ray • Can mimic biological conditions - p. H, temp, salt • Highly automated with more objective interpretation of data • information on dynamics • Quality indicators (resolution, R) • monitor conformational change on ligand binding • Surface residues and water molecules well defined • 2 structure derived from limited experimental data • Huge molecules and assemblies can be determined • need concentrated sample - lots of protein; aggregation issues • non-physiological conditions – crystallization difficult • size limited – ~40 k. Da for full structure determination • need heavy-atom derivatives – production not always trivial • more subjective interpretation of data • snap-shot of protein in time – less indication of mobility • lack of quality factors resolution and R-factor • flexible proteins difficult to crystallize

Identifying Unique NOEs • Filtered/Edited NOE: based on selection of NOEs from two molecules Identifying Unique NOEs • Filtered/Edited NOE: based on selection of NOEs from two molecules with unique labeling patterns 1 Unlabeled peptide H 1 H 13 C Labeled protein Only NOEs at the interface • Transferred NOE: Used for weak interactions (ligand in excess) and based on NOEs from bound state passed to free state H Only NOEs from bound state H H kon koff H