NMR structure calculation What Data How to get Structures Examples What Information What Software Precautions Kostas Tripsianes from data to structures •NMR experiments •Spectra analysis ■Sparky, NMRview, CARA, CcpNmr, Olivia •Resonance assignments •Backbone •Sidechain •Software: AUTOASSIGN, MARS, MBA, GARANT, PISTACCHIO •Structural restraints •NOE assignment / distances •torsion angles TALOS+M! •RDCs / orientation restraints •Structure calculations •Software: ARIA/CNS, CANDID/CYANA, XPLOR-NIH, UNIO •Structure validation •Software: iCing Why use NMR some proteins do not crystallize crystals do not diffract well, or at all can not solve the phase problem functional differences in crystal vs in solution can get information about dynamics NMR active nuclei Nucleus Nuclear spin Natural abundance Relative NMR sensitivity lH Vi 99.98% 100 *H 1 0.02% 0.96 »C V* 1.1% 1,6 ■sN v> 0.366% 0.1 :yF Vi 100% 833 3lp Vi 100% 6.6 Chemical shift Electrons around the nucleus shield it from the external magnetic field, the more electrons the weaker the field A y BBfl = (1 -o) B0 0) = Y (1 - o) B0 ö = (to — wrBl) / (ü0 x 106 Proton resonance spectrum Each proton atom in the molecule gives rise to a resonance line Chemical environment The chemical shift depends on the chemical environment RN H j aromatic 222 ac stylen : CH-'-J CH-Ar m □ CH, ZlCHlC =] a i-c=c 10 98765432 10 S( H)/ppm Chemical Shift Anisotropy /// I \\\ mm An important factor influencing the chemical shift are anisotropy effects, that are created by small additional fields Scalar or J-coupling Electrons in the bonds between the nuclei mediate an interaction, the scalar coupling Scalar or J-coupling Scalar coupling splits the signals according to the number of neighboring nuclei r,ch - ch, 480 400 320 240 I60 BO Scalar coupling contains structural information Dipolar coupling The nuclei interact directly through space via a dipole-dipole interaction In solution NMR this interaction is averaged to zero due to the fast isotropic movement of the molecules but it is still a source of relaxation NOE effect Inoe~ 1/r6 The intensity drops quickly with By increasing distance. The ' effect can only be observed up to 6 Angtrong Source of structural ^^^^ information!!! Challenges for determining protein structures using NMR •Proteins have thousands of signals •Assign the specific signal for each atom •Thousands of interactions between protons also need to be assigned •Need to transform the representation from spectra through interactions between atoms to spatial coordinates Hierarchical structure of proteins TAKE-HOME MESSAGE: function is derived from three-dimensional structure, and three-dimensional structure is specified by amino acid sequence ■ All natural proteins are constructed from the same set of 20 amino acids ■ Amino acids are classified based on the properties of R groups or sidechains hydrophobic, polar, charged ■ The primary structure (sequence) of a protein is the linear arrangement of the amino acids that compose it ■ The amino acids in a polypeptide are linked by peptide bonds planarity, hydrogen bonding potential, uncharged ■ The secondary structure refers to periodic structures stabilized by backbone hydrogen bonds a helix, p sheets ■ The tertiary structure refers to the overall conformation of a polypeptide (three-dimensional structure) hydrophobic interior, hydrophilic surface ■ Proteins consisting of more than one polypeptide chain display quaternary structure ■ The highest level of protein structure is the association of proteins into macromolecular assemblies Primary structure of proteins h/. h ft T T X I Repeating part: backbone Variable part: sidechain (R group) Most proteins contain 100 to 1000 residues Muscle protein titin consists of more than 27000 residues Secondary structure /3 sheets antiparallel parallel mixed 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. The twenty amino acids GLN '.HI CVS „ SEB Proton NMR A simple yet valuable NMR experiment Protons: natural abundance NMR active nuclei 10-50 u,M protein concentration Acquisition time seconds What is the value of this simple experiment? Is my protein folded? YES: structure possible NO: structure impossible-*magnificent disorder folded rc ■Wv/ folded Each proton in a protein resonates at a characteristic frequency on the NMR chemical shift scale, defined by its local structural environment. Illustrations of the Relationship Between MW, xc and T2 linewidth Av1w = 1MT„ MW 8 kDa Why multidimensional NMR? Advances of multidimensional NMR? The two major advantages of multidimensional NMR are: Improved resolution: Signals are spread over a surface (2D) or in a three-dimensional space (3D, 4D) Magnetization transfer: Signals result from the interaction between nuclei. That can be interactions through bond (via J-coupling) or through space (via NOE) Taken together this eases the interpretation and the assignment of the spectra considerably Homonuclear spectra (2D) Magnetization transfer between same nuclei. Both axes exhibit the chemical shift of the same type of nucleus. If a transfer has taken place, the signal has different frequencies in the two dimensions :cross peak If no transfer has taken place, the shifts are the same in both dimensions diagonal s diagonal signal Heteronuclear spectra (2D) Magnetization transfer different ^____Tronsfer H X nuclei. The two axes show the chemical shift of the respective type of nucleus. If a transfer has taken place, a signal appears at the intersection of the two frequencies, without a transfer there is no signal. 5H1 5H2 SH3 Resolving protons (13C/15N labeling) I I -•—Preparation—►«— Evolution —>•— Detection—► 2D NMR Spectrum t2 Fourier t1 Fourier Stacked Plot or Contour t1 Transform Transform MaP Correlations: Through-bond (J coupling) Through-space (dipolar coupling) 6) to "walk through" the backbone assignments • Requires a similar number of additional experiments to obtain the side-chain assignments The root spectrum 2D 1H-15N HSQC experiment • correlates backbone amide 15N through one-bond coupling to amide 1H • in principal, each amino acid in the protein sequence will exhibit one peak in the 1H-15N HSQC spectra . also contains side-chain NH2s (ASN,GLN) and NsH (Trp] i position in HSQC depends on local structure and sequence i no peaks for proline (no NH) Side-chain NH- INEPT Reverse INEPT lv "a.-í v a; / - n". „W "If ™ 8" * NMR Assignments 3D NMR Experiments * Consider a 3D experiment as a collection of 2D experiments i z-dimension is the 15N chemical shift • 1H-15N HSQC spectra is modulated to include correlation throui coupling to another backbone atom C|V-' 0 \ / C|S' -G.'-'-C'-1 ■ H • All the 3D triple resonance experiments are then related by the common 1H,15N chemical shifts of the HSQC spectra • The backbone assignments arethen obtained by piecing together all the "jigsaw" puzzles pieces from the various NMR experiments to reassemble the backbone NMR Assignments 3D NMR Experiments • Amide Strip 3D Strips can then be arranged in backbone sequential order to visual confirm assignments NMR Assignments 3D NMR Experiments • 3D HNCO Experiment i common nomenclature -> letters indicate the coupled backbone atoms i correlates NH1 to C_1 (carbonyl carbon, CO or C] no peaks for proline (no NH] • Like the 2D 1H-15N HSQC spectra, each amino acid should display a single peak in the 3D HNCO experiment i identifies potential overlap in 2D 1H-15N HSQC spectra, especially for larger MW proteins i most sensitive 3D triple resonance experiment CßH O C[V I , II >c . I N1-Ca'- M H H NMR Assignments 3D NMR Experiments • 3D HNCO Experiment 3DHNCO/HNC,\ i IHÍI i Air i II I I II *. Ill Í' 1* 1 III ft *I'-ÍM 1 Illl cv"c„ 1 I lllilf 1 c.,j,3c: 1 J G, i i íG, G, i K =4/_ 10; V = +/.,; rf,= y, (ij = (, 4>, = 2(1), : fc = »,-i + TPPl«1);»„ = *,K-»),,. & = !Amitt = UJfllů na (HNCOBNCA) NMR Assignments 3D NMR Experiments • 3D HN(CA)CO Experiment i correlates NH1 to Co1 and Co1"1 relays the transfer through Ca' without chemical shift evolution i contains both intra and sequential correlation i provides a means to sequential connect NH and CO chemical shifts > match NHi-COi (HNtCAlCOwrthNH-COi-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 i no peaks for proline (no NH) breaks assignment chain • but can identify residues i-lto prolines Cßh1 O Cß' -NM—CaM-CM—N'-Ca!- NMR Assignments 3D NMR Experiments • 3D HN(CA)CO Experiment 3D HN(CA)CO (HSQC) iulflf'1* aii-mi*..-,,,,-ii a if It if ifl 1 i, ».» "N III Ulxl III t'i 1 I III 13C 1 lllfl 1 iflll i 13C, 1 i h\Ua i 1 KG. (; 1 1 = +/-10; v • (i, = x; 0! = x, -x; <(>3 = x ; 4 = i = Mx), 4(-x> + TPPId,); , 4>m. = x = 5.4 ins, 7 = 11 ms. 8 = 6.8 ins, r| = 5.5 ins. CO connectivities •< n cp -ca- 0 II !c_-n1- cp ca- 0 ii c^ Asn i '1 ■ Asn 140 He 141 He 141 Ala 142 Ala 142 Glu 143 Slu 143 1 h | h" I 1 h" 1 1 i-1 1 1 HNCO i 1 cp 1 0 II cp l c 0 < n -ca- c-n - c°- c* 1 h 1 h" H 1 1 h" 1 - 1 i-1 1 1 i 1 HN CA-CB connectivities .cp! 0 cp 0 II l II 1 1 Asr\ 140 Asn140 He 141 He 141 flla 142 Ala 142 Glu 143 Slu 143 ^n-|ca-c-n]-ca-c^ 11 i I i I h ha |HJ h° 1 II 1 1 i-1 1 1 i 1 CBCA(CO)NH — --- C cp 0 cp 0 1 II 1 II -n-ca-c-n -c"-c-»- w1 h ha H ha — 1 i-1 1 '-i-1 H N HA connectivities cp o cp o I___ll__ I II N-[c«-c-Nl-ca-c-l i I i----i I i I 7^7 ' '-1- HBHA(CBCACO)NH cp o cp o I II I II n-cr^c-n -c--c-II II h Ha H Ha i-1 h h h ;n!40 Asn 140 He: Ha a 142 Ala 142 61u 143 Slu 143 HN Sequential assignments Starting points: Thr, Ser, Ala, Gly NMR Assignments 3D NMR Experiments • Backbone Assignments i The process is a multi-step approach: (1) correlate all the experimental data with each NH root observed in the 2D 'H-I5NHSQC spectra NMR Assignments 3D NMR Experiments • Backbone Assignments i The process is a multi-step approach: (2) Match pairs of NH roots based on i and i-1 correlations NMR Assignments 3D NMR Experiments • Backbone Assignments i The process is a multi-step approach: (3) Exlend pairs of NH roots and match to protein primary sequence Identify C overlapping J spin-system connect spin-systempairs NMR Assignments 3D NMR Experiments • Backbone Assignments i The process is a multi-step approach: (3) Extend pairs of Nil roots and match to protein primary sequence Identify possible residue types by chemical shift ranges NMR Assignments 3D NMR Experiments • Backbone Assignments i The process is a multi-step approach: (3) Extend pairs of NH roots and match to protein primary sequence 119. 65 5 B. 9 4 3.67 63.57 32.32 61.13 34.36 63.16 34.6B 43.71 32.27 Find potential match in sequence MTLKQ\|rV^RDDLICLSRGICLAVQVAHAAIIGYLICSDSSLRRICWLDBGQICICVVLICVICS LEELLGIK.H KA ES LG LVTGLVQ DAGLTEVPP GTITAV VIGP D EER K.I D K. VTGN L P L L K.L E HHHHHH Make assignment NMR Assignments Experiment Assignment Comment Relative sm pq HNCO <® kD. above use "H labeling 100 HMCA H(i).N(i).Ca(i),Cc((i-l) <20 kD. above use :H labeling 50/15 HN(CO)CA <20 kD. above use :H labeling 71 HNCAiLO B®,m C© <20 kD. above use "H labeling 13/4 CBCA(CO)XH H&MftC^i-liC^i-l) <20 kD. above use "H labeling 115 rap HBHA(CX>NH m,m H^i-D.Hpa-i) <30 kD. above use "H labeling B 9 rap CBCANH. HNCACB H(i).N(1),Ca(i).Cp(i), C^i-lXC^i-l) <15kD. above use "H labeling 41.7 a/Hi) 1.3/0.5 (HKX(COJNH-TOCSY Hii).N(i).Cal?il(i-l) <15-20 kD. above use "H labeling H(CCXCO)N"H-TOCSY H(L),M(i).HallFh(i-l) <15-20 kD. above use :H labeling HCCH-TOCSY ^alipli ^(ilijdi LI) ■.■II :■['.■.■ 1 in i■ ■ ■;1111 ■ -.in:.:-i\~nbnir with HCCONHtvpe experiments Sequence-Specific Assignment and powerful applications w : Sidechain assignments (1) a, -n^c—c • c —c- II o h- 8 — n I l" h - Ha I- - —c- II o NMR Assignments 3D NMR Experiments ' Side-chain Assignments . Help confirm the backbone assignment . Similar in principal to 2D assignment approach ► Correlate entire spin-system with NH backbone ► Use TOCSY to observe entire spin-system . CC(CO)NH & HCC(CO)NH - Relay magnetization from NH through side-chain carbon or hydrogen chemical shifts - Start simultaneously on all side-chain hydrogens -Also, overlap with Co. and Cp chemical shifts from other triple-resonance experiments to confirm side-chain assignments I- III i III i'r laiiitiiim ■Hi i í in III 1 lli.l.'i 11" nSoiařhí"*'! NMR Assignments Which II's match theC's? ■ IICC(CO)NII CC(CO)NII 3D NMR Experiments • Side-chain Assignments . CC(CO)MI & IICC(CO)NII i Can assign residue type by the number of observed resonances and the chemical shift ranges • may be able to assign Cy, C5, Cs from chemical shift values and from , previously assigned Ca and Cp • less likely to assignIly, 115 andlls; unless unique chemical shift • need companion experiments to connect carbon and hydrogen chemical shifts A20J E205 D206 D20T Biochemistry, Vol. 34, No. 42, 1995 Sidechain assignments (2) -n—c—c— I i II h h o This experiment correlates a 1H/13C pair to all other protons in the same aminoacid sidechain. Also, very useful for determining which 1H is directly attached to which 13C NMR Assignments 3D NMR Experiments ' Side-chain Assignments . HCCH-TOCSY& HCCH-COSY ► relays magnetization from side-chain and backbone 'H & ,3C via coupling constants ► Experiments have symmetry - 1Hot-13Ca diagonal shows cross peak to 'Hp AND - 'Hp-,3Cp diagonal shows cross peak to 'Ha. ► does not correlate to backbone NH-> no direct connection with other triple-resonance experiments - sample can be collected in D20 a) 3D HC(C)H-TOCSY 13C, I ~- f'«-N i i ifif F, 1 Ill fit t TPPl(tt); «j = 2(i). K-x)+ TPFllljl; (W= x. I NMR Assignments 3D NMR Experiments • Side-chain Assignments .HCCH-TOCSY Slices taken from different l3C chemical shift planes at different 'H chemical shifts illustrates the entire spin system for a single side-chain Symmetry - each HC5 shows a cross peak to Hct and the HCct shows a crosspeak to both HC8 Note: Symmetry peaks may notalwaysbe present (separate pathways, separate relative sensitivity). Presence of a symmetry peak increase the likelihood of correct assignment Journal of Bwmolecular NMR, 9 (1997) 445^446 NMR Assignments 4D NMR Experiments • Consider a 4D NMR experiment as a collection of 3D NMR experiments i still some ambiguities present when correlating multiple 3D triple-resonance experiments i 4D NMR experiments make definitive sequential correlations i increase in spectral resolution -Overlapis unlikely • loss of sensitivity an additional transfer step is required - relaxation takes place during each transfer rotameric states (VAL / LEU) Two chemically identical substituents on a tetrahedral carbon may be geometrically distinct Two such atoms are referred to as being prochiral. Designated as pro-R or pro-S based on same criteria as R and S fairest of all?" System in use at BioMagResBank (IUPAC/IUB Biochemistry 9, 3471-3479 [1970]) for VAL CG1=pro-R / CG2=pro-S for LEU CD1=pro-R / CD2=pro-S Switch off swapping when rotameric states are obtained (e.g. using 10% ,3C enriched medium) tautomeric state HIS 'H-15N long-range HSQC 9.0 8.5 8.0 7.5 7.0 6.5 (02-1H (ppm) nomenclature CYANA CNS HIS HIS+ HIST HISD HISE HIS ■r-bs. labeling schemes uniformly labeled Leu [17) pro-S methyl labeling (17 Leu + 3 Val] 24 26 B o 28 30 methyl labeling of Met (9) 32 2.0 1.5 1.0 0.5 0.0 -0.5 (U2-1H (ppm) NMR experiments • Backbone experiments CBCA(CO)NH S HNCACB 3D spectra HNCO S HN(CA)CO | |\ HAHB(CBCACO)NH S HN(CA)HA (4D, 5D ...) C(CCO)NH S HNHtocsy identify the spin-system • Sidechain experiments hCCH-TOCSY S HcCH-TOCSY (fold coupled carbon in both) • Aromatic rings CBHD CBHE but better rely on the noesy • Stereospecific assignments for VAL/LEU methyl groups 1H-13C HSQC on 10% labeled sample / pro-S labeling using an acetolactate precursor • Tautomeric states of histidines 1H-15N long-range HSQC • For larger proteins or complicated systems selective isotopic labeling of amino acids and stereospecific isotopic labeling of methyl group; • NOESY experiments HNH_noesy HCH_noesy obtain fold HCHaro_noesy CNH_noesy CCH_noesy optimize fold 2d_noesy homonuclear Nuclear Overhauser Effect (NOE) Distance: The strength of the dipolar interaction depends on the distance between the spins. The effect is inversely proportional to the sixth power of distance (1/r6) two spins, four energy levels dipolar interaction causes relaxation-induced transitions between any two levels