Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
G protein–coupled receptor wikipedia , lookup
Interactome wikipedia , lookup
Western blot wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Proteolysis wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Metalloprotein wikipedia , lookup
Resonance Assignment for Proteins Classical homonuclear (1H-1H) assignment methods: 1. Spin system assignments 2. Sequence-specific assignments 3. Sequential vs. Main-chain Directed Assignment Modern methods: Use of heteronuclear shift correlation, triple resonance experiments, etc. Resonance assignments •in order to be able to actually solve the structure of a protein, we first have to assign the spectrum •each peak corresponds to some proton within some amino acid residue. Is the sharp peak at -0.8 ppm a valine, leucine or isoleucine methyl? •even if we knew it was a valine methyl, which valine does it belong to? •even if we knew it was Val30, which of the two methyls is it? sequence of lysozyme: KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAA KFESNFNTQATNRNTDGSTDYGILQINSRWWCN DGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVS DGNGMNAWVAWRNRCKGTDVQAWIRGCRL Levels of resonance assignment • spin system assignment: is it Val, Ile or Leu? • sequence-specific assignment: is it Val 30 or Val 87? • stereospecific assignment: is it the pro-R or pro-S methyl of Val 87? Classical protein NMR: the basic plan In “classical” protein NMR, assignments are made by using 2-dimensional experiments to establish correlations between different 1H resonances. Recognition of characteristic patterns and networks of correlations then allows assignments to be made. Resonances are correlated either “through-bond”, mediated by the scalar coupling, or “through-space”, mediated by the spin dipolar coupling (nuclear Overhauser effect). H H through-bond (J-coupling) through-space (nOe) Basic features of 2D spectra HA HB 2Å chemical shift (ppm) HA HB diagonal peak: correlation of a resonance with itself 1H crosspeak: correlation of two different resonances by short interatomic distance or through-bond connection 1H chemical shift (ppm) Spin systems and scalar coupling networks • • a spin system is a set of 1H resonances connected (either directly or indirectly) by 1H-1H scalar couplings generally this means networks of 1H in which each 1H is connected to another member of the network by three or fewer covalent bonds-longer-range couplings are generally small, so experiments based on resonance correlation via scalar coupling will generally not detect fourand five-bond couplings Hc indirect connection H H geminal coupling (two-bond) J ~ -12 to -15 Hz H H vicinal coupling (three-bond) J ~ 2-14 Hz Ha Hb example of a spin system 2D COSY/TOCSY-->spin systems • • • • • COSY and TOCSY give crosspeaks when resonances are linked through scalar coupling COSY gives crosspeaks when 2and 3-bond couplings are present in TOCSY, longer range correlations are seen due to relays of 3-bond couplings these two techniques can be used to assign spin systems through recognition of coupling patterns recognition of the patterns at right also takes into account qualitative chemical shift information--the beta methyl of alanine, for instance, might be anywhere from ~0.9-1.7 but is never 3 or 4. o crosspeaks visible in COSY +, * crosspeaks visible in TOCSY Example of lysine spin system CO HN a b g d e NH3+ Ha He Hb Hd Hg Hd Hg Hb He Ha Sequence-specific assignments • • • • • suppose we have the sequence of our protein from some independent measurement suppose we’ve assigned an isoleucine spin system, and there’s only one isoleucine in the sequence (unique), at position 48. Then we know our isoleucine is Ile48. there won’t be very many unique amino acid residues in a protein, however. but there will be many unique dipeptide sequences but in order to use this fact, we need to be able to connect adjacent residues. unique residues (arrows) and unique dipeptide sequences in lac repressor Linking spin systems using nOe’s • because the nOe depends upon interatomic distance and not upon J coupling, it can be used to connect spin systems which are adjacent in space but not part of the same spin system, for instance two residues adjacent in the sequence •general nomenclature for interatomic distance between atoms A and B in residues i and j: dAB(i,j) • nOe correlations are denoted using the distance nomenclature, e.g. “dbN(i,i+1) nOe” or “dbN (i,i+1) correlation” • daN(i,i+1), dNN(i,i+1), and sometimes dbN(i,i+1) are used to connect adjacent residues 2D NOESY: linking spin systems 4.HN/5.HN 5.HN/6.HN diagonal: no magnetization transferred crosspeaks: intersection of chemical shifts of atoms which are close in space, i.e. < 5 Å 1H 6.HN/7.HN 3.HN/4.HN 1H portion of 2D NOESY of P22 cro showing dNN(i,i+1) correlations-can “walk” along the chain from one residue to the next. Residues 3-7 shown. Sequential assignment • the technique of making the spin-system assignments, followed by sequence-specific assignment using unique fragments of sequence, is known as sequential assignment (Wuthrich) • there are alternatives to this protocol: one is known as main-chain directed assignment (Englander). This technique does not focus on assigning all the spin systems first. Rather, it focuses on the backbone and links sizable stretches of backbone residues via sequential (i,i+1) nOe’s and other nOe’s that are characteristic of secondary structures. This technique is particularly useful when there is some knowledge of secondary structure beforehand. Close interatomic distances in secondary structures parallel beta-sheet antiparallel beta-sheet alpha-helix type I turn type II turn Close interatomic distances in 2ndary structures you’ll often see nOe’s associated with secondary structure charted in this way: residue # •a thick bar means a strong nOe (short distance), a thin bar means a weak nOe (long but still visible distance) • these sorts of charts allow one to make secondary structure assignments more or less concurrently with sequential assignments. As we will see, coupling constants and chemical shifts also aid in secondary structure assignment ...you can see that it would be easiest to link adjacent residues in helices with sequential amide-amide nOe’s, whereas in beta sheets (strand) sequential alpha-amide nOe’s are stronger d~2.8 Å d~2.2 Å Summary of main-chain directed approach 1. assign a few unique spin systems and use as entries onto the backbone Arg Tyr Ser Ala Ala Asn Trp 3. fill in missing spin system assignments 2. walk down the backbone using sequential and other backbone nOe’s “backbone” refers to alpha and amide protons Summary of sequential approach 1. assign most or all spin systems Arg Tyr Ser Ala Ala Asn Trp 3. assemble larger sections of sequence-specific assignments from dipeptide fragments, until the whole protein has been assigned 2. connect adjacent spin systems using backbone nOe’s to identify unique dipeptides “backbone” refers to alpha and amide protons Assignment methods that use heteronuclear shift correlation • • • • for larger proteins (>10-15 kD), assignment methods based on 2D homonuclear 1H-1H correlation methods (COSY/TOCSY/NOESY) don’t work very well because of overlapping resonances and broad linewidths. an alternative (which is now used even for small proteins in most cases) is to use heteronuclear shift correlation experiments on 13C, 15N labelled samples. in these experiments, magnetization is transferred from 1H to 13C and/or 15N through large one-bond scalar couplings. Some relevant scalar coupling constants: 15N-1H HSQC based techniques •as we have seen, one of the simplest types of heteronuclear shift correlation is the HSQC experiment, which correlates 1H chemical shift to the chemical shift of a 15N or 13C connected by a single bond •heteronuclear shift correlation can be combined with homonuclear experiments such as 1H-1H NOESY or TOCSY to yield 3-dimensional spectra 3D HSQC-NOESY and HSQC-TOCSY these planes can be thought of as a 15N-1H HSQC these planes can be thought of as a 1H-1H NOESY the 15N shift dimension can resolve peaks that would overlap in a 2D NOESY view of a 3D NOESY experiment Triple-resonance experiments • • • • there is a whole raft of experiments that use both 13C and 15N correlations to 1H nuclei the beauty of these experiments is that they can connect adjacent residues without requiring any nOe information-it’s all through-bond scalar coupling interactions. Makes sequence-specific assignment more reliable. they also use mostly one-bond couplings, which aren’t very sensitive to the protein conformation (unlike, say, three-bond couplings, which vary significantly with conformation, as we will see) limiting factors: 13C is expensive and these exp’ts can be tricky