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NMR in biology: Structure, dynamics and energetics Gaya Amarasinghe, Ph.D. Department of Pathology and Immunology CSRB 7752.

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Presentation on theme: "NMR in biology: Structure, dynamics and energetics Gaya Amarasinghe, Ph.D. Department of Pathology and Immunology CSRB 7752."— Presentation transcript:

1 NMR in biology: Structure, dynamics and energetics Gaya Amarasinghe, Ph.D. Department of Pathology and Immunology CSRB 7752

2 NMR? Nuclear Magnetic Resonance Spectroscopy Today, we will look at how NMR can provide insight in to biological macromolecules. This information often compliment those obtained from other structural methods.

3 /1dnmr.htm NMR Spectra contains a lot of useful information: from small molecule to macromolecule. nature00860_F1.html Few peaks Sharper lines Overall very easy to interpret Many peaks Broader lines Overall NOT very easy to interpret

4 Structure determination by NMR NMR relaxation– how to look at molecular motion (dynamics by NMR) Ligand binding by NMR – Energetics

5 Outline for Bio 5068 December 11 Why study NMR (general discussion) 1.What is the NMR signal (some theory) 2.What information can you get from NMR (structure, dynamics, and energetic from chemical shifts, coupling (spin and dipolar), relaxation—next class) 3.What are the differences between signal from NMR vs x-ray crystallography (we will come back to this after going through how to determine structures by NMR) Practical aspects of NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample preparation (very basic aspects & deal with specific labeling during the description of experiments) Assignments and structure determination 1.2-D experiments 2.3/4-D experiments 3.Restraints and structure calculations Assessing quality of structures 1.NMR structure quality assessment 2.Comparison with x-ray

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8 Nuclear transitions Rotational transitions Translational transitions Electronic transitions Diffractions NMR works in the rf range- after absorption of energy by nuclei, dissipation of energy and the time it takes Reveals information about the conformation and structure. For diffraction, the limit of resolution is ½ wavelength!!

9 Protein Structures from an NMR Perspective Background – We are using NMR Information to “FOLD” the Protein. – We need to know how this NMR data relates to a protein structure. – We need to know the specific details of properly folded protein structures to verify the accuracy of our own structures. – We need to know how to determine what NMR experiments are required. – We need to know how to use the NMR data to calculate a protein structure. – We need to know how to use the protein structure to understand biological function

10 Protein Structures from an NMR Perspective Distance from Correct Structure NMR Data Analysis Correct structure X Not A Direct Path! Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold Initial rapid convergence to approximate correct fold Iterative “guesses” allow “correct” fold to emerge Analyzing NMR Data is a Non-Trivial Task! there is an abundance of data that needs to be interpreted

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12 Current PDB statistics (as of 3/27/2012) Exp.Met hod Proteins Nucleic Acids Protein/Nucleic Acid Complexes Total X-RAY NMR ratio

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14 Nuclei are positively charged many have a spin associated with them. Moving charge—produces a magnetic field that has a magnetic moment Spin angular moment

15 MassChargeI Even I=0 EvenOdd I= integer OddI=half integer

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18 How do we detect the NMR signal?

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21 Next time—pick up on chemical shifts

22 Practical aspects of NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample preparation (very basic aspects & deal with specific labeling during the description of experiments)

23 Practical aspects of NMR 1.instrumentation 2.Sample signal vs water signal 3.Sample preparation (very basic aspects & deal with specific labeling during the description of experiments) ion/NMSU_NMR300_J.html

24 Sample preparation using recombinant methods

25 Vinarov et al., Nature Methods - 1, (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics

26 Segment labeling can simplify NMR spectra Native chemical ligationExpressed protein ligation Muir et al. Curr Opin Biotechnol.Muir et al. Curr Opin Biotechnol Aug;13(4):

27 Sample requirements and sensitivity Methyl groups are more sensitive than isolated Ha spins Source :

28 Sample requirements and sensitivity Cryoprobes are 3-4 times better S/N than standard probes (2x in high salt) Source :  M not mM!!

29 Why use NMR ?  Some proteins do not crystallize (unstructured, multidomain)  crystals do not diffract well  can not solve the phase problem  Functional differences in crystal vs in solution  can get information about dynamics

30 Protein Structures from an NMR Perspective Overview of Some Basic Structural Principals: a)Primary Structure: the amino acid sequence arranged from the amino (N) terminus to the carboxyl (C) terminus  polypeptide chain b)Secondary Structure: regular arrangements of the backbone of the polypeptide chain without reference to the side chain types or conformation c)Tertiary Structure: the three-dimensional folding of the polypeptide chain to assemble the different secondary structure elements in a particular arrangement in space. d)Quaternary Structure: Complexes of 2 or more polypeptide chains held together by noncovalent forces but in precise ratios and with a precise three-dimensional configuration.

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33 Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment State II – Conformational restraints Stage III – Calculate and refine structure

34 Resonance assignment strategies by NMR

35 Illustrations of the Relationship Between MW,  c and T 2

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43 NMR Assignments 3D NMR Experiments 2D 1 H- 15 N HSQC experiment correlates backbone amide 15 N through one-bond coupling to amide 1 H in principal, each amino acid in the protein sequence will exhibit one peak in the 1 H- 15 N HSQC spectra  also contains side-chain NH 2 s (ASN,GLN) and N  H (Trp)  position in HSQC depends on local structure and sequence  no peaks for proline (no NH) Side-chain NH 2

44 3D NMR Experiments Consider a 3D experiment as a collection of 2D experiments  z-dimension is the 15 N chemical shift 1 H- 15 N HSQC spectra is modulated to include correlation through coupling to a another backbone atom All the 3D triple resonance experiments are then related by the common 1 H, 15 N chemical shifts of the HSQC spectra The backbone assignments are then obtained by piecing together all the “jigsaw” puzzles pieces from the various NMR experiments to reassemble the backbone NMR Assignments

45 3D NMR Experiments Amide Strip 3D cube 2D plane amide strip Strips can then be arranged in backbone sequential order to visual confirm assignments

46 NMR Assignments 3D NMR Experiments 3D HNCO Experiment  common nomenclature  letters indicate the coupled backbone atoms  correlates NH i to C i-1 (carbonyl carbon, CO or C’)  no peaks for proline (no NH) Like the 2D 1 H- 15 N HSQC spectra, each amino acid should display a single peak in the 3D HNCO experiment  identifies potential overlap in 2D 1 H- 15 N HSQC spectra, especially for larger MW proteins  most sensitive 3D triple resonsnce experiment  may observe side-chain correlations 1 J NC’ 1 J NH

47 NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment  correlates NH i to C  i  relays the transfer through C  i without chemical shift evolution   uses stronger one-bond coupling  contains only intra correlation  provides a means to sequential connect NH and C  chemical shifts  match NH i -CO i (HN(CA)CO with NH i -CO i-1 (HNCO)  not sufficient to complete backbone assignments because of overlap and missing information  every possible correlation is not observed  need 2-3 connecting inter and intra correlations for unambiguous assignments  no peaks for proline (no NH) breaks assignment chain  but can identify residues i-1to prolines 1 J C  C’ 1 J NH 1 J NC 

48 NMR Assignments 3D NMR Experiments 3D HN(CA)CO Experiment Amide “Strips” from the 3D HNCO and HN(CA)CO experiments arranged in sequential order HNCO and HN(CA)CO pair for one residues NH Connects HN i -CO i with HN i -CO i-1 Journal of Biomolecular NMR, 9 (1997) 11–24

49 NMR Assignments 4D NMR Experiments Consider a 4D NMR experiment as a collection of 3D NMR experiments  still some ambiguities present when correlating multiple 3D triple-resonance experiments  4D NMR experiments make definitive sequential correlations  increase in spectral resolution – Overlap is unlikely  loss of digital resolution – need to collect less data points for the 3D experiment – If 3D experiment took 2.5 days, then each 4D time point would be a multiple of 2.5 days i.e. 32 complex points in A-dimension would require an 80 day experiment  loss of sensitivity – an additional transfer step is required – relaxation takes place during each transfer Get less data that is less ambiguous?

50 NMR Assignments

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52 Why use deuteration? What are the advantages? What are the disadvantages?

53 2D 15 N-NH HSQC spectrum of the 30 kDa N-terminal domain of Enzyme I from the E. coli Effects of Deuterium Labeling only 15 N labeled 15 N, 2 H labeled Current Opinion in Structural Biology 1999, 9:594–601

54 Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment State II – Conformational restraints Stage III – Calculate and refine structure

55 NMR Structure Determination With The NMR Assignments and Molecular Modeling Tools in Hand: All we need are the experimental constraints  Distance constraints between atoms is the primary structure determination factor.  Dihedral angles are also an important structural constraint What Structural Information is available from an NMR spectra? How is it Obtained? How is it Interpreted?

56 4.1Å 2.9Å NOE CHCHCHCH NH NH CHCHCHCH J NOE - a through space correlation (<5Å) - distance constraint Coupling Constant (J) - through bond correlation - dihedral angle constraint Chemical Shift - very sensitive to local changes in environment in environment - dihedral angle constraint Dipolar coupling constants (D) - bond vector orientation relative to magnetic field to magnetic field - alignment with bicelles or viruses D NMR Structure Determination

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60 Protein Secondary Structure and Carbon Chemical Shifts 11 22 33 44 II  II  III  IV

61 NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts TALOS + Shen et al. (2009) J. Biomol NMR 44:213

62 NMR Structure Determination Protein Secondary Structure and Carbon Chemical Shifts TALOS+  Given the C , C  Chemical shift assignments and primary sequence  Compares the secondary chemical shifts against database of chemical shifts and associated high-resolution structure  comparison based on “triplet” of amino acid sequences present in database structures with similar chemical shifts and secondary structure  Provides potential ,  backbone torsion constraints  Issues: May not provide a unique solution, two or more sets of  are possible  Can not initially use TALOS results if ambiguous. Can add constraint latter if consistent with structure.

63 NMR Structure Determination Protein Secondary Structure and 3J HN  Karplus relationship between  and 3J HN    =180o  3J HN   ~8-10 Hz   -strand   = -60o  3J HN  = ~3-4 Hz   -helix Vuister & Bax (1993) J. Am.Chem. Soc. 115:7772

64 NMR Structure Determination Protein Secondary Structure and 3J HN  Karplus relationship between  and 3J HN   Measure 3J HN  for a protein using HNHA  Ratio of cross-peak to diagonal intensity yields coupling constant  Common approach to measure coupling constants in complex protein NMR spectra J. Am. Chem. Soc. 1993,115,

65 Protein Structure Determination by NMR Stage I—Sequence specific resonance assignment State II – Conformational restraints Stage III – Calculate and refine structure

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67 Protein Structures from an NMR Perspective What Information Do We Know at the Start of Determining A Protein Structure By NMR? Effectively Everything We have Discussed to this Point!  The primary amino acid sequence of the protein of interest. ► All the known properties and geometry associated with each amino acid and peptide bond within the protein. ► General NMR data and trends for the unstructured (random coiled) amino acids in the protein.  The number and location of disulphide bonds. ► Not Necessary  can be deduced from structure.

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73 Double the nOe restraints From above

74 7 restraints/residue 10 restraints/residue 13 restraints/residue 16 restraints/residue

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76 Wüthrich et al., J. Virol. February 15, 2009; 83:

77 Analysis of the Quality of NMR Protein Structures With A Structure Calculated From Your NMR Data, How Do You Determine the Accuracy and Quality of the Structure? Consistency with Known Protein Structural Parameters  bond lengths, bond angles, dihedral angles, VDW interactions, etc  all the structural details discussed at length in the beginning Consistency with the Experimental DATA  distance constraints, dihedral constraints, RDCs, chemical shifts, coupling constants  all the data used to calculate the structure Consistency Between Multiple Structures Calculated with the Same Experimental DATA Overlay of 30 NMR Structures

78 Analysis of the Quality of NMR Protein Structures As We have seen before, the Quality of X-ray Structures can be monitored by an R-factor No comparable function for NMR Requires a more exhaustive analysis of NMR structures

79 Analysis of the Quality of NMR Protein Structures Root-Mean Square Distance (RMSD) Analysis of Protein Structures A very common approach to asses the quality of NMR structures and to determine the relative difference between structures is to calculate an rmsd  an rmsd is a measure of the distance separation between equivalent atoms  two identical structures will have an rmsd of 0Å  the larger the rmsd the more dissimilar the structures 0.43 ± 0.06 Å for the backbone atoms 0.81 ± 0.09 Å for all atoms

80 Analysis of the Quality of NMR Protein Structures Root-Mean Square Distance (RMSD) Analysis of Protein Structures A variety of approaches can be used to measure an RMSD  only backbone atoms  exclude disordered regions  only regions with defined secondary structure  only the protein’s active-site region  on a per-atom or per-residue basis rmsd difference between NMR and X-ray structure

81 Analysis of the Quality of NMR Protein Structures Is the “Average” NMR Structure a Real Structure? No-it is a distorted structure  level of distortions depends on the similarity between the structures in the ensemble  provides a means to measure the variability in atom positions between an ensemble of structures Expanded View of an “Average” Structure Some very long, stretched bonds Position of atoms are so scrambled the graphics program does not know which atoms to draw bonds between Some regions of the structure can appear relatively normal

82 Analysis of the Quality of NMR Protein Structures As We Discussed Before, PROCHECK is a Very Valuable Tool For Accessing The Quality of a Protein Structure ► Correct ,     distribution ► Comparison of main chain and side-chain parameters to standard values

83 Analysis of the Quality of NMR Protein Structures NMR R-factor difference between expected and observed NOEs  expected NOEs  structure  observed NOEs  NMR spectra  also includes unassigned NOEs  perfect fit would yield R = 0 R-factors have not been readily adapted in NMR community  affected by completeness of assignments, peak overlap, sensitivity, noise, extent of data (RDCs, coupling constants, etc  trends with rmsd without complications Journal of Biomolecular NMR, 17: 137–151, 2000.

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85 Protein Structures from an NMR Perspective Distance from Correct Structure NMR Data Analysis Correct structure X Not A Direct Path! Interpreting NMR Data Requires Making Informed “Guesses” to Move Toward the “Correct” Fold Initial rapid convergence to approximate correct fold Iterative “guesses” allow “correct” fold to emerge Analyzing NMR Data is a Non-Trivial Task! there is an abundance of data that needs to be interpreted

86 Timescales of Protein Motion N H Energy landscape and dynamics high energy barriers = slow rate low energy barriers = fast rate

87 Why do proteins move? Broad, shallow energy potential – Thermal energy is sufficient for the protein to sample many different conformations Change in conditions – Interaction with a small molecule or binding partner, change in temperature, ion concentration, etc. – Now a different conformation is lower in energy Sequence encodes both protein structure and protein flexibility – Non-bonded interactions determine the lowest energy conformation(s) Sequence Stability Flexibility Function Function requires Stability: the right chemical and spatial features in the right place to bind ligand, catalyze a chemical reaction, etc. Flexibility: the ability to move in order to control access in and out of the active site and to provide energy for chemical reactions

88 NMR Analysis of Protein Dynamics Hydrogen-Deuterium Exchange As we saw before, slow exchanging NHs allowed us to identify NHs involved in hydrogen-bonds. Similarly, slow exchanging NHs are protected from the solvent and imply low dynamic regions. Fast exchanging NHs are accesible to the solvent and imply dynamic residues, especially if not solvent exposed. Protein sample is exchanged into D 2 O and the disappearance of NHs peaks in a 2D 1 H- 15 NH spectra is monitored. Protein Science (1995), 4:

89 NMR Analysis of Protein Dynamics Hydrogen-Deuterium Exchange The observed NH intensity loss can be fit to a simple exponential to measure an exchange rate (k ex ) These exchange rates may range from minutes to months!  NHs with long exchange rates indicate stable or low mobility regions of the protein  NHs with short exchange rates indicate regions of high mobility in the protein

90 NMR Analysis of Protein Dynamics Hydrogen-Deuterium Exchange As expected, majority of NHs that exhibit slow exchange rates are located in secondary structures fast exchanging NHs are located in loops, N- and C-terminal regions

91 NMR Parameters for Protein Dynamics Number of signals per atom Line-widths Hydrogen Exchange (H-D) Hetero-nuclear { 15 N, 13 C} Relaxation measurements – T 1 (spin-lattice relaxation time) – T 2 (spin-spin relaxation time) – Hetero-nuclear NOE

92 NMR Relaxation  After an RF pulse system needs to relax back to equilibrium condition  Related to molecular dynamics of system  may take seconds to minutes to fully recovery  usually occurs exponentially: – (n-n e ) t displacement from equilibrium value n e at time t – (n-n e ) 0 at time zero  Relaxation can be characterized by a time T – relaxation rate (R): 1/T  No spontaneous reemission of photons to relax down to ground state  Two types of NMR relaxation processes  spin-lattice or longitudinal relaxation (T 1 )  spin-spin or transverse relaxation (T 2 ) B 1 off… (or off-resonance) MoMo z B1B1 z x M xy yy 11 MoMo y z x T 1 & T 2 relaxation

93  Spin-lattices or longitudinal relaxation  Relaxation process occurs along z-axis  transfer of energy to the lattice or solvent material  coupling of nuclei magnetic field with magnetic fields created by the ensemble of vibrational and rotational motion of the lattice or solvent.  results in a minimal temperature increase in sample  Relaxation time (T 1 )  exponential decay NMR Relaxation M z = M 0 (1-exp(-t/T 1 ))

94 T 2 relaxation NMR Relaxation  Spin-Spin or Transverse relaxation  Relaxation process in the x,y plane  Related to peak line-width – Inhomogeneity of magnet also contributes to peak width  T 2 may be equal to T 1, or differ by orders of magnitude – T 2 can not be longer than T 1  No energy change ( derived from Heisenberg uncertainty principal)

95 NMR Relaxation Mechanism for Spin-lattices and Spin-Spin relaxation Illustration of the Relationship Between MW,  c and T 2

96 Conformational Exchange Increases the Rate of Transverse Relaxation (R 2 ) in NMR Spectra R 2 = R R ex R ex depends on: Kinetics: k ex = k A + k B Thermodynamics: p A *p B Structure: 

97 NMR Analysis of Protein Dynamics k – exchange rate – peak frequency h – peak-width at half-height e – with exchange o – no exchange k =  (h e -h o ) k =  (  o 2 -  e 2 ) 1/2 /2 1/2 k =   o / 2 1/2 k =  o 2 /2(h e - h o )

98 PreparationRelaxationFrequency LabelingAcquisition In the Absence of Chemical Exchange Magnetization Refocuses Following a 180° Pulse PreparationFrequency LabelingAcquisition

99 PreparationRelaxationFrequency LabelingAcquisition Relaxation Due to Chemical Exchange Leads to Loss of Transverse Magnetization No Chemical Exchange With Chemical Exchange

100 PreparationRelaxationFrequency LabelingAcquisition Increasing the Number of CPMG Pulses Can Recover Magnetization Due to R ex R ex R20R20 For 2-state exchange in the ms-µs regime, quantitative analysis can in principle yield: p A, p B, k A, k B,  ······

101 Summary --- NMR relaxation/dynamics High sensitivity and site specific information may need isotopic labeling May require assignment of resonances Can help narrow construct space and identify interfaces regions that interact with solvent or binding partners

102 NMR Analysis of Protein-Ligand Interactions NMR Monitors the Different Physical Properties That Exist Between a Protein and a Ligand

103 NMR Analysis of Protein-Ligand Interactions Ligand Line-Width (T 2 ) Changes Upon Protein Binding As we have seen before, line-width is directly related to apparent MW  a small-molecule (~100-1,000Da) is orders of magnitude lighter than a typical protein (10s of KDa)  a small molecule has sharp NMR line-widths (few Hz at most))  protein has broad line-widths (10s of Hz)  if a small molecule binds a protein, its line-width will resemble the larger MW protein + Small molecule: Sharp NMR lines Broad NMR lines Sharp NMR lines Broad NMR lines  c  MW/2400 (ns)

104 Slow isomerization of dimethyl amino group at low temperature produces distinct signals for each methyl At increasing temperatures (faster exchange rates) peaks broaden and eventually coalesce into one average signal Chemical exchange NMR timescales For binding reactions, slow exchange (higher affinity) produces distinct signals for free and bound states at intermediate titration points - follow binding reaction by watching bound/free peak intensities grow/diminish Fast exchange - only one set of peaks throughout titration, shifting in proportion to changing ratio of free:bound

105 Summary --- NMR ligand binding High sensitivity and site specific information may need isotopic labeling May require assignment of resonances Affinity measurements are only valid for low affinity interactions Complex structures can be determined for high affinity interactions

106 Comparison of NMR and X-ray Structures

107 NMR and X-ray Structures Comparison of NMR and X-ray Structures Science (2000) 289, large ribosomal subunit X-ray structure There is no theoretical limit to the size of the structure that can be determined by X-ray crystallography. Requires a crystal that diffracts! - requires highly pure samples - requires high solubility (~mM) - requires high stability (crystal may take weeks to months to form) - requires absence of aggregation/ppt - may requires seleno-Met labeling for phase determination - usually need to test 100s to 1,000s of crystal conditions - requires a protein that will form a crystal (may require site-directed mutant, N-,C- terminal truncation or using sequences from different species)

108 NMR and X-ray Structures Comparison of NMR and X-ray Structures where: r = radius k = Boltzman constant  = viscosity coefficient Conversely, there is a molecular-weight upper limit for NMR structures.  molecular-weight of a protein is related to its radius which in turn is related to the protein’s rotational correlation time (  c ) :  rotational correlation time (  c ) is the time it takes a molecule to rotate one radian (360 o /2  ).  the larger the molecule the slower it moves   c is related to the efficiency of T 2 relaxation

109 NMR and X-ray Structures Comparison of NMR and X-ray Structures As we have seen to this point, that an NMR structure is determined indirectly by combining NMR experimental data as target functions with traditional geometrical potential energy functions. Conversely, an X-ray structure is determined by directly fitting the structure against the electron density maps. This approach still uses XPLOR to refine the structure and maintain proper geometry (bond lengths, bond angles)

110 NMR and X-ray Structures Comparison of NMR and X-ray Structures As a result, a single optimal structure can be determined to represent the experimental X- ray data where the r-factor indicates the quality of the fit and the data indicates the resolution of the structure Conversely, the NMR data can be equally represented by an ensemble of structures and there currently is no corresponding equivalent to the r-factor or resolution The EMBO Journal (2000) 19(13) 3179 Biochemistry (2000) 39(31),

111 The resolution of the structure is the minimum separation of two groups in the electron-density plot that can be distinguished from one another. NMR and X-ray Structures Comparison of NMR and X-ray Structures Resolution increases (d) as you move out concentric circles in the X-ray diffraction pattern Acta Cryst. (2000). D56, 1015–1016 Example of Ultra-High Resolution X-ray Diffraction Pattern Bragg equation: 2dsin  =n X    d X Note: diffraction intensity decreases as you move to outer circle

112 NMR and X-ray Structures Comparison of NMR and X-ray Structures Protein Science (1996). 5: NMR and X-ray structures generally exhibit the same fold Local differences may be attributed to: 1) dynamics 2) crystal-packing interactions 3) solid vs. solution state - solvent is present in crystals - lowest energy conformer in crystal? 4) Resolution/experimental error Nevertheless, there are some examples where distinct functional differences are observed between the NMR and X-ray structures

113 NMR and X-ray Structures Comparison of NMR and X-ray Structures Illustration of the large differences between the NMR (blue) and X-ray (red) structures of the Ca 2+ –calmodulin complex “The difference between the crystal and solution structures of Ca 2+ –calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets.” Nature Structural Biology (2001), 8(11), X-ray structure suggested a “dumb-bell” structure with an extended  -helix NMR structure indicated the central helix was unstructured and dynamic.

114 NMR and X-ray Structures Comparison of NMR and X-ray Structures Protein Dynamics Is Routinely Measured From NMR Data Dynamic Data Is Also Implied From the X-ray B- Factor (temperature factor in the PDB). Overall Poor Correlation Between NMR Dynamic Data and B-factors 1) dynamic regions may have low B-factors if stabilized by an interaction not present in solution 2) low dynamic regions may have high B- factors due to resolution issues not related to dynamics – various crystal contacts, lack of uniformity in crystals, etc.

115 Final thoughts?

116 “NMR of Proteins and Nucleic Acids” Kurt Wuthrich “Protein NMR Spectroscopy: Principals and Practice” John Cavanagh, Arthur Palmer, Nicholas J. Skelton, Wayne Fairbrother “Principles of Protein Structure” G. E. Schulz & R. H. Schirmer “Introduction to Protein Structure” C. Branden & J. Tooze “Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis” R. Copeland “Biophysical Chemistry” Parts I to III, C. Cantor & P. Schimmel “Principles of Nuclei Acid Structure” W. Saenger Some Other Recommended Resources

117 Some Important Web Sites: RCSB Protein Data Bank (PDB)Database of NMR & X-ray Structures BMRB (BioMagResBank)Database of NMR resonance assignments CATH Protein Structure ClassificationClassification of All Proteins in PDB SCOP: Structural Classification of Proteins Classification of All Structures into Families, Super Families etc. DALICompares 3D-Stuctures of Proteins to Determine Structural Similarities of New Structures NMR Information ServerNMR Groups, News, Links, Conferences, Jobs NMR Knowledge Base A lot of useful NMR links

118 Many slides have been either taken directly or adapted from the following sources: protein-structures-university-nebraska-lincoln-324/ David Cistola (Wash U) Kevin Gardner/Carlos Amzcua (UTSW) Or as cited


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