Class Note for CHEM 534B at UA
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Date Created: 02/06/15
quotImproving Protein Structure Determination by NMR using Partial Orientation in Solutionquot Neil E Jacobsen PhD NMR Facility Manager Department of Chemistry University of Arizona BCP Journal Club Thursday Sepmmber 30 2004 Residual Dipolar Couplings in Protein Structure Determination The old way of protein structure by NMR 0 Local vs Global information in mapping 0 Measureables in solution state NMR 0 Dipolar Coupling D vs Scalar Coupling J 0 Methods for partial alignment of proteins 0 How can we use this new information 0 Applications to protein structure problems 0 Your New Toys the Varian Inova 6OO NMR Protein Structure Determination Assign all NMR Peaks to Specific Positions Measure HH Distances Less than 5 A Measure NHCaH Dihedral Angles Align Secondary Structure Elements Determine Tertiary Structure by Minimizing Errors in Distances and Angles Surveying 0n the Angstrom Scale C IT H C Distance H T T C ICiQ H H 30 Classical Method for Protein Structure Determination by NMR Measure Distances between Protons if lt 5 A Classical Method for Protein Structure Determination by NMR Assignments Name each Proton Classical Method for Protein Structure Determination by NMR Structure Calculation Minimize Distance Errors MAJOR U SV CITIES Limitations of the Classical Approach Historically a serious limitation of NMR structure determination has been the inability of NMR to determine global conformations of macromolecules While NMR can precisely determine local structural features errors tend to propagate through the length of the molecule Thus if there are no NOE interactions between separate parts of a molecule the long range conformation becomes poorly defined Hansen Mueller and Pardi Nature Structural Biology 5 1998 1065 1074 Classical Method Small NOE Distance Errors Accumulate and Lead to Poor Orientation of Distant Domains MAJOR U SV CITIES Classical Method Plus RDC s Residual Dipolar Couplings Provide a North Star Orientation Reference Classical Method Plus RDC s Residual Dipolar Couplings Provide a North Star Orientation Reference MAJOR U SV CITIES Refinement with RDC s Residual Dipolar Coupling Restraints Give Accurate Orientation of Distant Domains MAJOR U SV CITIES Measurables of NMR in Solution Chemical Shifts Exact Resonant Frequency of each Nucleus H C N This gives an address to uniquely study each one O gtgt J Couplings Scalar Couplings For HXCH This Gives the H to H l Dihedral Angle NOE s Nuclear Overhauser Effect as Intensity of this interaction between H s gives distance measurement Chemical Shift The resonant frequency Peak position in spectrum resonant frequency v y B frequency is proportional to magnetic field felt by the nucleus The magnet supplies a field BO which is slightly modified by the bonding electrons around the nucleus Glycine N C z CO 839 397 ppm Chemical Shifts of Amino Acids in Disordered Peptides Each amino acid has characteristic chemical shifts for its HN Ha H6 H7 etc positions giving it a recognizable fingerprint in the 1H spectrum Ala 7 gt u B Met u 7 vz gd Cys 7 77 a 7 15 Asn gt oc gt ii ASP 13 7 Pro 39 5 1amp7 Glu oz B 7 Gin 7 a 7 TB Phe oc B 7 Arg 7 11 a 417 Gly 7 e u Ser 7 u quot His gt u 5 Thr 7 uB y lie 7 7 0177 41713551 7 Val u 7 A57 777 Lys 7 a gt Y 7 Trp 7 gt 0 p 7 Leu a 57 6 Tyr 7 a p 5 0 4 0 3 0 2 0 1 0 5 0 4 0 3 0 2 0 1 0 Protein Chemical Shifts are Modified by the Local Environment OLhellx sheet nearby aromatic rings shift left or right H Chemical Shins RandomCoil vs Heregulin EGF Domain llN I II l I aromatic I methyl I 90 30 0 60 50 40 30 20 10 ppm a Glycine 0 o 17 or I G18 l I I 539 I I G42 I I I Alanine I I niA l l I A43 I n I A59 I l I Leucine o n a I L3 t m u liLZG I quotI I 7L33 n In I I L62 I I II Spinspin Splitting or SpinSpin Coupling 0 1H magnet can be aligned with at state or against 3 state the magnetic eld 0 The bonded 15N or 13C nucleus shifts its resonance frequency slightly to higher or lower frequency as a result J N1 1 S Ha HB 50 50 I 15N frequency The Console Digital and Radio Frequency Electronics The Driver s Seat Your New Toys Varian Inova 600 MHZ Spectrometer installed in September 2004 Four Channels 1H 13C 15N 2H Cryogenic Probe to be installed in November 2005 Total Cost 1000000 Biochemistry and IBSB Located in Old Chemistry 121D Current Research and New Applications of RDCS Global Fold Determination from RDCs alone Structural Genomics Simultaneous Assignment and Structure Determination from RDCs using Chemical Shifts Dynamics of N H bond mobility from reduction of RDCs Small molecule stereochemistry and bound ligand orientation Relative Orientation of Two NH vectors Without knowing the orientation of the alignment frame in the molecular frame pairwise angular restraints can be generated Skrynnikov and Kay J Biomol NMR 18 2000 239252 Probability MaltodextrinBinding Protein 42 kD Histogram of 443 onebond dipolar couplings 015 El L 1E6 NH C N CaC most likely f igrw l Exmrimmml DC listmram Narrow bars are theoretical values based on Aa 155 X 103 R 019 Skrynnikov and Kay J Biomol NMR 18 2000 239252 Assuming that all orientations are equally sampled in the measured D values Da and R can be obtained from the histogram Histogram of Measured Dipolar Couplings Number of Measured Dipolal Couplings 21 D2I1 32R quotDal 32R Value of Residual Dipolar Coupling Hz D D3 3 cos2 1 3R sin2 cos2CD lapping the N H Vector in the Alignment Frame z Dzz gt D gt DXX MM If you know Da y 9 and R you can orient the NH z D vector D DXX gives the largest range of possible orientations so it should be the most commonly found valueom I 0 90 180 270 360 Dxx gt D gt DW Alignment of Two Domains B and C domains of barley lectin Fischer Losonczi Weaver Prestegard Biochem 1999 38 90139022 X Wheat Germ Agglutinin X Ray Barley Lectin same homologous protein alignment frame for two domains Rubredoxin 54 residues assembled from six fragments each separated by a single Proline Translation connects the aligned fragments Tian Valafar and Prestegard J Am Chem Soc 2001 123 11791 Ag r A A Fitting of D values within a molecular fragment 0 If the structure of one domain one element of secondary structure eg helix or even one peptide plane is known five or more D values within the fragment can be fitted to locate the alignment axes 0 Doing this for a number of domains allows the domains to be aligned relative to each other since there is only one alignment frame Use of Two Different Alignment Media to Resolve Ambiguity Streptococcal Protein G 56 Residues X B 7 Da R 71 2155 77 066 forfd at pH 54 66 89 22 97 023 for bicelles at pH 70 Clore Statich and Gronenborn J Am Chem Soc 1998 120 10571 2 NH vector in two alignment frames Structure Refinement Using RDCS Rat apo 100BBB Ca2 binding EF hand gt 500 RDC restraints backbone RMSD 104 9 029 A Residues in favored regions of Ramachandran 76 9 86 Poorly defined 3rd helix now precise change in helix 3 to helix 4 angle is 900 upon Ca2 binding not 1160 Drohat Tjandra Baldisseri and Weber Protein Sci 8 1999 800809 0 Yeast Splicing Factor Prp40 are the two adjacent WW domains positioned to allow simultaneous binding of poly proline binding partners Backbone RMSD 114 9 055 A Each WW domain leans away from the linker region so the two binding sites face in opposite directions Wiesner Stier Sattler and Macias J Mol Biol 324 2002 807822 Incorporating RDC S into Traditional Structure Calculations l A rough initial structure is generated using NOE s and dihedral angles or from homologous Xray 2 Fitting of a subset of RDC s leads to alignment parameters and axes 3 RDC s are predicted and a pseudoenergy term is added to the total energy Erdc krdc Dealc Dobs2 4 Continue energy minimization and return to step 2 Finding the Alignment Parameters and Axes 0 Given a trial structure and at least 5 RDC values you can solve linear equations ie fit 0 Da and R can be estimated from a histogram of all measured RDC values 0 Determine only the relative angles between pairs of NH or CH vectors Paradox You need a structure to determine a structure 0 You need to have a structure with a molecular coordinate system 0 You need to find five unknowns 1 Da the degree of alignment 2 R the rhombicity 3 The orientation of the alignment axes Relative to the molecular frame or 3 and y Calculated D Hz Predicted Residual Dipolar Couplings Compared to Measured Values 0 Human Ubiquitin Predicted Values based on the 18 A Xray structure Optimization of the fit gave Da and R Three angles which orient the alignment frame relative to the Xray coordinate system or 3 and y Euler angles Tjandra and Bax Science 278 1997 4 1 o 2 Measured D Hz 11111114 Measured 1H15N Coupling is Sum of Scalar J and Dipolar D 116 0 Peak Separation JD J is nearly constant 90 Hz quot185 0 D is positive N or negative and less than I 120 PPm Bax and Tjandra J Biomol NMR 10 1997 289292 Predicting the Value of the Residual Dipolar Coupling D 0 In the Alignment Frame Polar coordinates can be used to describe the orientation of the N H vector D Da 3cos2 1 32 Rsin2 cos2613 2 t0 1 1 to 1 where Da 13 DZZ DXX Dyy2 is a measure of the degree of alignment and R 13 DXX Dyy D81 is a measure of the rhombicity deviation from aXial symmetry The Alignment Frame within the Molecule Three orthogonal axes The long z axis lt3cos2 Z1gt DZZ The medium x axis lt3cos2 X1gt DXX The short y axis lt3cos2 y1gt DW 2 Z V Lx Ly O 1x Side View Side View Top View Alignment in Mechanically Stressed Gels Pores are deformed into ellipsoids leading to weak and tunable alignment of solutes Bicelles Bilayered Mixed Micelles 31 ratio Dimyristoylphosphatidylcholine C14 to Dihexanoylphosphatidylcholine C6 Coin shaped 40 nm and aligned with the magnetic field Can switch from isotropic lt 27C to liquid crystal gt 27C Degree of alignment proportional to lipid conc 10393 to 10394 free DHPC edge DHPC 39 30 12 DMPC and sum DHPC Methods For Inducing a Slight Orientational Preference in Solution 0 Use the Inherent Magnetic Properties of the Molecule Paramagnetic Herne BDNA naturally align With the field 0 Liquid Crystals Bicelles Filamentous Phage Particles Stretched or Compressed Polyacrylamide Gels Partial Orientation Gives NonZero Average of Dipolar Coupling Probability Sphere is Stretched to an Ellipsoid by 21 15 sin 3cosz 1 05 0 05 1 215 2 25 3 quot 35 o5 o oo 90 o o o o 15 G in radians sum 3702 Dipo for ar Couplings Average to Zero Rapid Isotropic Reorientation Average of 300s2 1 over all NH orientations is zero if all are equally probable equally distributed over sphere 08 06 04 02 0 O O2 04 O6 O8 1 12 sin 3cosz 1 9 390 0 0 039 I O O 9 O 4 0 05 25 3180 3 G in radians sum 390390105 Effect of 1H Magnet 0n the Field Experienced by 15N Distance r is fixed by N NH bond length N 9 9 9 N I S All nuclear magnets o are aligned with the s external field 6 N 395 1H magnet can be either 9 up or down 0 o 0 02 Effect of 1H is modulated by the angle 9 with the external field freq shift 3cos2 1 Orientation of NH vector with respect to the magnetic field The angle 9 can be determined by another NMR measureable the Residual Dipolar Coupling RDC Two Kinds of SpinSpin Coupling Scalar or ThroughBond Coupling J Relatively Small 1H15N Coupling 90 Hz Does Not Depend on Orientation of N H Dipolar or ThroughSpace Coupling D Very Large 1H15N Coupling 25000 Hz Depends on Orientation of N H 3cos2 1 Averages to Zero for Rapid Isotropic Motion Inserting the Sampm Larisa Yeomans NMR Facility RA
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