Resonance assignments in proteins. Christina Redfield

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1 Resonance assignments in proteins Christina Redfield 1. Introduction The assignment of resonances in the complex NMR spectrum of a protein is the first step in any study of protein structure, function or dynamics. Prior to 1980, assignment was achieved using 1D NMR, and based, for the most part, on the assumption that the structure of the protein in solution was the same as in the X-ray structure. The introduction of 2D NMR techniques such as COSY and NOESY in the early 1980's dramatically increased the resolution of protein NMR spectra. This led to the development of a systematic method for the assignment of the 2D NMR spectra of proteins that relied only on information about the amino acid sequence of the protein; this method is the sequential assignment procedure. The availability of uniform 15 N labelling and the development of 3D NMR methods in the late 1980 s led to increased resolution in NMR spectra and increased the size of proteins that could be assigned using the sequential assignment methodology. Double labelling with 15 N and 13 C led, in the early 1990 s to the development of an alternative assignment approach based on scalar couplings; this again increased the molecular weight limit for complete assignment. Recently, this triple-resonance approach in conjunction with deuteration and new TROSY methods has increased the molecular weight limit to beyond 30kD. In this chapter the elements of the sequential assignment and the triple-resonance assignment strategies are outlined. 2. Sequential assignment method for 1 H NMR spectra The sequential assignment method consists of two stages. The first involves identification of systems of spin-spin coupled resonances which belong to a particular type of amino acid. This is achieved using COSY, RELAY and TOCSY experiments. During this process the spin systems of all the alanine residues may be identified but there is nothing to distinguish one alanine spin system from another. The second stage involves assignment of each spin system to a particular residue in the amino acid sequence. This can not be achieved using the COSY-type experiments because there is no resolved 1 H- 1 H spin-spin coupling across the peptide bond. Instead assignments are deduced from the through-space dipole-dipole information found in NOESY spectra. The procedures used in this two-stage assignment method are described below. 2.1 Assessing the quality of 1 H 2D NMR spectra Before attempting sequential assignment using 1 H NMR methods it is advisable to determine whether the protein of interest will give 2D NMR spectra of the required quality. The coupled H N and H α resonances of each residue should give rise to a cross peak in the fingerprint region of the COSY spectrum (F 2 ~10.5 to 6.5 ppm, F 1 ~6.0 to 2.5 ppm). In addition, cross peaks arising from the side chain H N 's of Arg and Lys may be observed in this region of the spectrum (F 2 ~7.5 to 6.5 ppm, F 1 ~3.5 to 2.5 ppm). If more than ~10% of the expected cross peaks are missing then the steps outlined in Protocol 1 should be taken in order to determine the reason for the absence of peaks and to overcome the problem before continuing with the assignment process. Protocol 1. Reasons for loss of H N -H α cross peaks 1. Cross peaks are missing because of saturation of H α resonances beneath the water resonance. This can be overcome using pre-tocsy COSY or SCUBA-COSY methods. Alternatively, collect the spectrum at 10 o C above or below the previous temperature; the H 2 O resonance shifts significantly with temperature whereas most H α resonances do not. 1

2 2. H N intensity is decreased due to cross saturation from the H 2 O signal because H N s are exchanging with H 2 O. This problem can be identified by comparing the intensity of the H N region in 1D 1-1 spectra collected with and without presaturation of the water. If the intensity of the H N 's differs in these experiments then cross saturation is a problem. Spectra should be collected with 1-1 or pulsed-field gradient methods instead of solvent presaturation. Alternatively, H N exchange rates can be decreased by adjusting the sample ph closer to the ph minimum for hydrogen exchange (ph ~4) or by decreasing the sample temperature. 3. Cross peaks may be below the signal to noise level because of a small H N -H α coupling constant (J) or a large line width (LW). Cancellation of cross-peak intensity occurs because of overlap of antiphase components when J is less than or equal to the line width. Collect a TOCSY spectrum with a short isotropic mixing period and compare the number of H N -H α cross peaks to that seen in the COSY spectrum. If there are additional peaks in the TOCSY spectrum then these may correspond to residues where J < LW. Line widths can be decreased by increasing the temperature. If this is feasible then collect another COSY spectrum and look for additional peaks. If the protein does not give close to the expected number of COSY peaks then the protein is not a good candidate for sequential assignment using 1 H 2D techniques alone. Alternative methods based on 15 N or 15 N/ 13 C labelling should be considered. If the COSY spectrum is of acceptable quality then the first stage of the sequential assignment process can be started Stage 1: Spin system identification The first stage of sequential assignment involves the identification of systems of spin-spin coupled resonances corresponding to individual amino acid residues. The random coil chemical shift values for resonances in the common amino acids are shown in Figure 1 (3). Many of the amino acids have unique spin system topologies and will give rise to unique patterns of cross peaks in a COSY spectrum (4). If there is no overlap in the spectrum then spin systems can readily be identified from the COSY spectrum alone. However, in most proteins there is some degree of overlap that is likely to increase with the size of the protein. When overlap occurs unambiguous spin system assignments are not usually possible on the basis of the COSY spectrum alone and other 2D datasets are required. It will be shown later that the analysis of NOE data for the second stage of sequential assignment relies most heavily on the H N and H α resonances. Therefore, the approach to spin system identification described here emphasises the COSY H N -H α peak. Information about the type of residue from which a particular H N -H α cross peak arises is collected using a variety of 2D experiments. For many residues, such as alanine and threonine, complete spin system identification is quite straightforward even for large proteins. For other residues, including arginine and lysine, complete spin system identification is more difficult. However, the lack of complete spin systems for these residues does not necessarily hamper the sequential assignment process. Spin system information can be obtained from two types of experiment. The first includes COSY, DQF COSY, RELAY and double RELAY. The cross peaks in these spectra are characterised by a pattern of antiphase fine structure that reflects the active and passive coupling constants of the spin system. This pattern can provide useful information for assignment that is usually not available from TOCSY spectra. The second is the TOCSY experiment that is widely used for several reasons. Firstly, the in-phase nature of the cross peaks leads to signal-to-noise ratios that may be better than would be observed in COSY-type spectra where cancellation of intensity occurs in the antiphase cross peaks. Secondly, by adjusting the isotropic mixing period used in the experiment long-range connectivities in the spin system can be observed; in favourable cases complete spin systems can be identified even for Arg and Lys residues Spin system information from COSY, RELAY and double-relay spectra Before attempting to identify the side chain resonances associated with each H N -H α cross peak it is useful to analyse the information available in the fingerprint region of the COSY spectrum. Part of this 2

3 region of COSY spectra collected with 512 and 256 t 1 values are compared in Figure 2. The cross peaks shown in Figure 2a have various shapes and cross peak fine structures. The cross peaks composed of eight antiphase components are characteristic of the glycine spin system which has two H α coupled to H N. The square four component cross peaks arise from spin systems in which both H α -H β coupling constants or the single H α -H β coupling constant are small (< 5 Hz). The rectangular four component peaks arise from spin systems in which there is at least one large H α -H β coupling constant (> 9Hz) or two intermediate H α -H β coupling constants (6-8 Hz). Thus, the H N -H α cross peak shape can provide information about H α -H β coupling constants (5). This information cannot readily be obtained from the COSY spectrum shown in Figures 2b; the loss of information results from the poor digital resolution in F Glycine spin system identification As discussed above the three coupled protons of the glycine spin system form an AMX spin system which gives H N -H α cross peaks with a characterisitic 8 component shape. As a result glycine spin systems are usually easy to identify as shown in Protocol 2. Protocol 2. Glycine spin system identification 1. Identify a pair of cross peaks in an H 2 O COSY, with the characteristic 8-component peak shape, which correlate a H N chemical shift with two H α shifts. Confirm that these two H α belong to a single spin system by identifying a cross peak correlating the two H α resonances in a D 2 O COSY spectrum. 2. The remaining 8-component H N -H α cross peaks may correspond to glycines in which the second H N -H α cross peak is too weak to observe due to a small H N -H α coupling constant. This second peak should be observed in RELAY or TOCSY spectra. Confirm that these two H α belong to the same spin system as in step A glycine with degenerate H α resonances will have a single H N -H α peak with only 4 components that looks like the peaks arising from other residues. Wait until the stage 2 NOE analysis to identify these glycines Alanine, threonine, valine, isoleucine and leucine spin system identification The spin systems of alanine, threonine, valine, isoleucine and leucine residues all contain an H α, H β or H γ coupled to one or two methyl groups. The H N -H α fingerprint peaks of these residues can be assigned to one of these amino acid types if a connection can be established between the H N or H α and the CH 3 group(s). COSY, RELAY and double-relay peaks correlating the H N or H α to the CH 3 group can be distinguished from other cross peaks in the same regions of the spectrum, arising from Arg, Lys and Pro, on the basis of their strong intensity and narrow peak shape in the CH 3 dimension, as shown in Figure 3. The intensity of the RELAY and double-relay peaks depends on the H N -H α and H α -H β coupling constants. The procedure for identifying these spin systems is described in Protocol 3. Protocol 3. Ala, Thr, Val, Ile and Leu spin system identification 1. Ala: Look for RELAY peaks from H N to Ala H β. Confirm the Ala spin systems by identifying strong H α -H β cross peaks in the COSY spectrum. 2. Thr: Look for double-relay peaks from H N to Thr H γ. Look for RELAY peaks from Thr H N to H β and from H α to H γ, and for COSY peaks from H α to H β and from H β to H γ to confirm the Thr spin systems. 3. Val: Look for pairs of double-relay peaks from H N to Val H γ. Look for RELAY peaks from Val H N to H β and for pairs of RELAY peaks from Val H α to H γ. Look for COSY peaks from H α to H β and pairs of COSY peaks from H β to H γ to confirm the Val spin systems. 4. Ile: Look for double-relay peaks from H N to Ile H γ2. Look for RELAY peaks from Ile H N to H β and from Ile H α to H γ2. Look for COSY peaks from H α to H β and from H β to H γ2 to confirm the Ile spin 3

4 systems. The connectivity between the Ile H γ1 -H δ and the Ile H N -H α -H β -H γ2 subsystems can sometimes be made on the basis of H γ1 -H γ2 and H β -H δ RELAY peaks. 5. Leu: Look for pairs of double-relay peaks from H α to Leu H δ. These are usually only seen for small proteins or peptides Type J spin system identification The remaining amino acids can be divided into those with protons at the γ position and those without. The latter group includes Asn, Asp, Cys, His, Phe, Ser, Tyr and Trp. These residues have H β resonances usually downfield of ~2.5 ppm (Fig. 1) and will be denoted as type J.* (* The letters J, U and X are chosen to denote certain amino acid categories because these letters are not used in the one letter amino acid code.) These amino acids all have identical H N -H α -H β spin subsystems because there is no resolved coupling between the H β protons and any protons beyond the γ position. The H α and H β protons of these eight residues form an AMX system. The H α -H β coupling constants depend on the torsion angle χ 1 and determine the cross peak fine structure observed for the H α -H β and H β -H β cross peaks of these residues as well as influencing the shape of the H N -H α cross peak (see 2.2.1). The procedure for identifying these spin systems is summarised in Protocol 4. Protocol 4. Type J spin system identification 1. Look for pairs of H α -H β COSY cross peaks with a common H α shift and H β shifts downfield of ~2.5 ppm. Confirm that the H α -H β peaks arise from the same residue by locating the H β -H β cross peak. One strong and one weak peak indicates one large and one small H α -H β coupling constant (χ 1 = -60 o or 180 o ). A pair of weak peaks indicates two small H α -H β coupling constants (χ 1 =60 o ). A pair of strong peaks indicates intermediate H α -H β coupling constants indicative of averaging about χ Look for single H α -H β COSY cross peaks with H β downfield of ~2.5 ppm. If all Gly, Thr, Ile and Val cross peaks have been identified then these single peaks arise from type J residues. This peak is likely to arise from a large H α -H β coupling constant; the peak arising from the small coupling constant is weak and has disappeared into the noise. Look for a strong H α -H β RELAY peak to the missing H β position. Confirm that the two H β arise from the same residue by locating the H β -H β cross peak. 3. For each H α -H β spin system identify all the possible H N -H α peaks with the correct H α shift. Try to identify the correct H N -H α peak using peak shape and RELAY effects. (a) If the spin system was characterised by one large and one small H α -H β coupling constant then the correct H N -H α cross peak should have a rectangular shape. A H N -H β RELAY peak should be observed to the H β resonance with the large H α -H β coupling constant. (b) If the spin system was characterised by two small H α -H β coupling constants then the correct H N -H α cross peak should have a square shape. H N -H β RELAY peaks may not be observed because of the small H α -H β coupling constants. (c) If the spin system was characterised by two intermediate coupling constants then the correct H N -H α cross peak should have a rectangular shape. RELAY peaks may be observed to both H β positions. Some of the type-j residues have protons beyond the γ position. If these additional protons can be correlated with the H N -H α -H β -H β subsystem then a more specific residue-type assignment within the type-j category can be made. These correlations must be based on NOE effects because no resolved longrange coupling exists between the H β and H δ protons. The 4 aromatic type-j amino acids, His, Phe, Tyr and Trp, have unique patterns of cross peaks in the region of ~6-8 ppm. For Tyr and Phe an NOE effect is observed between H δ and H β. NOE effects between the H N, H α and H δ may also be observed depending on the residue conformation (φ, χ 1, χ 2 ). For Trp an NOE effect is observed between either H δ1 or H ε3 and H β. For His an NOE effect is observed between H δ2 and H β for some values of χ 2. 4

5 Type U spin system identification The final group of amino acid residues includes Lys, Arg, Met, Gln, Glu and Pro. All of these residues have two protons at the γ position that are coupled to H β. These residues have H β resonances upfield of ~2.2 ppm (Figure 1) and are designated type U. The backbone protons of leucine residues for which H N to H δ or H α to H δ connectivities have not been established also fall into this category. Residues within the type U category can sometimes be further divided on the basis of the chemical shifts of H γ as shown in Figure 1. The procedure for identifying the type U spin systems is summarised in Protocol 5. Protocol 5. Type U spin system identification 1. Look for H α -H β COSY cross peaks with H β upfield of ~2.2 ppm; peaks should be elongated along the H β dimension (Fig. 3). The side chains of these residues are often on the surface of the protein and consequently are more likely to undergo averaging about χ 1 and χ 2. Therefore, two strong H α -H β cross peaks with coupling constants of ~6.5 Hz are often observed. 2. For each H α -H β spin system identify all possible H N -H α peaks with the correct H α chemical shifts. Spin systems which give one or two strong H α -H β COSY peaks will have a rectangular H N -H α peak. 3. Look for H N -H β RELAY peaks for each of the possible fingerprint region peaks. These RELAY peaks should be elongated in the H β dimension compared to Ala H N -H β RELAY peaks. 4. Look for H α -H γ RELAY and H N -H γ double-relay peaks. If H γ is downfield of H β the spin system is likely to arise from Glu, Gln or Met. If H γ has a similar chemical shift to or is upfield of H β the spin system is likely to arise from Lys, Arg, Leu or Pro. If resonances beyond the γ position can be identified then the spin system can be assigned specifically to one of the 7 type U residues. Otherwise, the backbone spin system is assigned to a more general category and the identification of side chain protons can be made after the sequential assignments are completed Type X cross peak category Once Protocols 2-5 have been carried out a large number of spin systems should have been identified. At this stage it should be possible to label each peak in the fingerprint region with a one-letter amino acid identifier. For cross peaks corresponding to Gly, Ala or Thr this identifier will be the specific one letter amino acid code, G, A or T. For other cross peaks corresponding to residues such as Ser, Arg or Met this identifier will correspond to a more general group of amino acids such as type J or type U. It is likely that there will be some H N -H α cross peaks without an identifier at this point, and these peaks can be labelled type X. Most of these peaks will have the square shape characteristic of small H α -H β coupling constants; it is these small J values which lead to the absence of observable COSY and RELAY peaks Obtaining spin system information from TOCSY spectra The TOCSY experiment uses isotropic mixing to transfer magnetisation between scalar coupled spins within an amino acid residue. The amount of spin system information contained in a TOCSY spectrum will depend on the length of the TOCSY mixing time, the size of the coupling constants in the spin system, and resonance linewidths. With shorter TOCSY mixing times (20-25 ms) only H N -H α peaks may be observed. As the mixing time is increased (up to ~100 ms) connectivities to H β, H γ and other sidechain protons will appear. In general the intensity of H N -H α peaks will depend on the 3 J HNHα coupling constant; an intense peak is seen in the case of a large coupling constant and a weaker peak for a small coupling constant. Residues with a large H N -H α coupling constant will give stronger intensity H N -H β connectivities than residues with small H N -H α coupling constants. Although the amount of spin system information will, in principle, increase as the TOCSY mixing time is increased, transverse relaxation will result in a decrease in signal at very long mixing times. Spin system information is usually obtained from 5

6 a series of TOCSY spectra collected with increasing mixing times. As a general rule, longer-range connectivities, such as H N -H δ, will only appear in TOCSY spectra once the shorter-range connectivities such as H N -H β have appeared. However, different behaviour may be observed for the same amino acid type at different positions in the sequence as a consequence of differences in coupling constants. For example, a Val residue with large H N -H α and H α -H β coupling constants will show H N -H γ peaks in a TOCSY spectrum collected with a mixing time of 50 ms while a Val residue with small coupling constants may only show an H N -H α peak in this spectrum. Rules for interpreting the spin system information contained in TOCSY spectra are summarised in Protocol 2. Protocol 6: Rules for analysing TOCSY spectra 1. Collect 2 or more TOCSY spectra with mixing times ranging from 20 to 100 ms. For each fingerprint H N -H α peak make a list of the TOCSY peaks that are observed with increasing mixing time. 2. If a single peak is observed upfield of ~1.75 ppm at all mixing times then the spin system is likely to be an Ala. The peak should have a narrow shape in the CH 3 dimension. 3. If 2 peaks are observed downfield of 3.5 ppm then the spin system is a Gly, Ser or Thr. If 3 peaks are observed then the spin system is a Ser. If 2 peaks are observed downfield of 3.5 ppm and 1 peak is observed upfield of ~1.6 ppm then the spin system is a Thr. 4. If 1 or 2 peaks are observed in the region of ppm and no peaks are observed further upfield then the spin system is either an Asp, Asn, Cys, His, Phe, Trp or Tyr and can be classified at type J. 5. If at least 1 peak is observed upfield of ~2.25 ppm then the spin system is either Arg, Gln, Glu, Ile, Leu, Lys, Met or Val and can be classified as type U. (a) If an additional peak is observed downfield of this peak at longer mixing times then the spin system is likely to be Glu, Gln or Met. (b) If peaks are observed upfield of ~1.5 ppm with a narrow shape then the spin system is likely to be Ile, Leu or Val. If more than 3 peaks upfield of 2.5 ppm are observed then the spin system is Ile or Leu. (c) If more than 2 peaks are observed between 1.5 and 2.25 ppm the spin system is likely to be Arg or Lys. At long mixing times 1 or 2 peaks at ~3 ppm may be observed. A total of 6 and 8 peaks in addition to the H N -H α peaks are expected for Arg and Lys, respectively. 6. If no peaks are observed other than the H N -H α peak then the spin system must be classified at type X. 2.3 Stage 2: Sequence specific assignment The second stage of assignment involves the assignment of an amino acid spin system identified in stage 1 to a specific residue in the protein sequence. This is achieved by correlating an amino acid spin system with the spin systems of its neighbouring residues in the sequence, and relies on the through-space connectivities observed in NOESY spectra. In the early 1980's Wuthrich and coworkers showed that, for all sterically allowed values of φ, ψ and χ 1 at least one of the distances between H N, H α and H β of adjacent residues is short enough to give rise to an observable NOE effect (1,2,4). The most useful NOE effects for sequential assignment were found to involve the H α of residue i and the H N of residue i+1, d αn (i,i+1), the H N 's of residues i and i+1, d NN (i,i+1), and the H β of residue i and the H N of residue i+1, d βn (i,i+1). The intensities of these NOE effects depend on the torsion angles ψ, φ and ψ, and χ 1 and ψ, respectively. This torsion angle dependence means that specific types of secondary structure are characterised by specific sequential NOE effects. In the extended backbone structure, characteristic of β-sheet, the distance d αn (i,i+1) is short, 2.2 A, whereas d NN (i,i+1) is longer, 4.3 A. In helical structure the distance d NN (i,i+1) is short, 2.8 A, whereas d αn (i,i+1) is longer, 3.5 A (4). NOE effects between H N, H α and H β resonances are not, however, restricted to adjacent residues of the sequence. Longer range NOE effects involving these protons are also used to identify regions of secondary and tertiary structure. Wuthrich and coworkers have shown that if both a d αn and a d NN NOE or a d αn and a d βn NOE effect are observed between two residues then in >90% of cases these two residues are adjacent in the sequence if an upper limit of 3.8 A is used for the observation of an NOE effect. 6

7 Thus, the identification of two of the three NOE effects is a more reliable criterion for sequential assignment (1,2,4). The second stage of assignment involves the identification of stretches of sequential NOE effects. In practice a full set of NOE connectivities from the N to C termini is not observed. Breaks in the sequential assignment occur for several reasons. First, a sequential d NN (i,i+1) NOE will not be resolved from the diagonal if the two H N resonances have very similar H N chemical shifts. Second, a break may occur if the COSY H N -H α cross peak of a residue does not appear. Third, a break in the sequential assignment can occur at each proline residue. For these reasons the sequential assignment process is carried out in shorter peptide segments within the protein sequence. If a strong d αn NOE is observed between two spin systems then these are likely to be adjacent in the sequence. If the resonances of the spin systems have been assigned to a specific amino acid type, such as Gly and Ala, then sequential assignment may be possible from this single NOE effect because the pair G-A may occur only once in the sequence. However, as the size of the protein increases the occurrence of unique pairs of residues in the sequence decreases. Thus, it may be necessary to identify the residue adjacent to the Gly or Ala before a specific assignment can be made. If the resonances of the spin systems have been assigned to a more general class of amino acids such as type J or type U, or if no information about the spin system is known, type X, then sequential assignment of the basis of the single NOE is very unlikely. Additional NOE's to the adjacent residues will be needed before an assignment can be made. The procedure for carrying out the second stage of assignment for proteins of this kind follows. The only part of the NOESY spectrum that needs to be considered for sequential assignment is the section with H N chemical shifts in F 2 and the entire chemical shift range in F 1. The first step in the analysis involves the identification of NOE effects involving aromatic resonances. These can be identified by overlaying the NOESY spectrum of fully exchanged protein in D 2 O and the spectrum of the protein in H 2 O. The peaks between 10.5 and 6.5 ppm along F 2 that occur in both spectra involve aromatic protons rather than H N 's and are not used in sequential assignment. These peaks should be labelled in the H 2 O spectrum. Intraresidue NOE effects can be identified by overlaying the fingerprint region of the COSY (or TOCSY) and NOESY spectra in H 2 O. For each H N -H α cross peak in the COSY label the corresponding intraresidue H N -H α peak in the NOESY. In addition label other intraresidue peaks, identified in stage 1, in the NOESY spectrum. The unlabelled NOE peaks that remain in the H N -H N and H α -H N regions of the NOESY spectrum will correspond to interresidue NOE effects. Further analysis of the NOESY spectrum does not require overlaying of the NOESY and COSY spectra because the intraresidue NOE effects have now been labelled in the NOESY spectrum. The next stage in the analysis involves the identification of the sequential NOE effects between a H N -H α cross peak i and its adjacent residues i-1 and i+1. Depending on the nature of the secondary structure present these adjacent residues can be identified on the basis of d αn or d NN NOE connectivities. The procedure for identification of adjacent residues is illustrated in Figure 4 and described in detail in Protocol 7. Protocol 7. Identification of adjacent residues from d NN, d αn and d βn NOE effects 1. Start with most downfield H N in the spectrum and locate its d αn (i,i) peak in the NOESY spectrum. 2. Look along the H N (i) shift for a strong d αn (i-1,i) interresidue NOE peak. If a peak is found then look along the H α shift of this peak (i-1) for an intraresidue d αn (i-1,i-1) NOE. 3. Look along the H α (i) shift for a strong d αn (i,i+1) interresidue NOE peak. If a peak is found then look along the H N shift of this peak (i+1) for an intraresidue d αn (i+1,i+1) NOE. 4. If a strong d αn NOE effect other than d αn (i,i) is not observed for this H N then look along the H N (i) shift for two medium intensity d NN NOE peaks. If two peaks are observed then these are likely to correspond to residues i-1 and i+1. Look along the H N (i-1) and H N (i+1) shifts for weak intraresidue d αn NOE peaks. In order to distinguish the H N -H α peak of residue i-1 from that of i+1 look along the H N (i) shift for a weak d αn (i-1,i) interresidue NOE peak to one of the two possible H α shifts. The H α shift observed corresponds to residue i-1. Look along the remaining H N (i+1) shift for a weak d αn (i,i+1) interresidue NOE effect to 7

8 the H α of residue i. 5. If a strong d αn (i-1,i) NOE effect was observed in step 2 but no strong d αn (i,i+1) effect was observed in step 3 then look along the H N (i) shift for a d NN (i,i+1) NOE peak. Look along the H N (i+1) shift for a weak intraresidue d α N(i+1,i+1) NOE peak and a weak interresidue d αn (i,i+1) NOE peak to the H α of residue i. 6. If a strong d αn (i,i+1) NOE was observed in step 3 but no strong d αn (i-1,i) effect was observed in step 2 then look along the H N (i) shift for a d NN (i-1,i) NOE peak. Look along the H N (i-1) shift for a weak intraresidue d αn (i-1,i-1) NOE peak. Look along H N (i) for a weak interresidue d αn (i-1,i) NOE peak to the H α of residue i Confirm that the H N -H α peaks i-1, i and i+1, identified in steps 2-6, arise from adjacent residues by identifying d βn (i-1,i) and d βn (i,i+1) NOE effects. 8. Repeat steps 2 to 7 for the next intraresidue d αn (i,i) peak in the NOESY spectrum. 9. Make a list of each H N -H α COSY peak and the COSY peaks that correspond to the two adjacent residues. Also record the amino acid identifier for each H N -H α peak. This list of adjacent spin systems is now used as the basis for making sequence specific assignments. If the spin system identification was carried out as described above then each fingerprint region peak will have an identifier such as G, A, T, J, U or X. If very complete spin system identification was achieved then specific identifiers such as Y, N, R and Q will also be found. The spin systems that have been assigned to one specific type of amino acid residue are now used as the basis for assignment because short sequences containing these residues are more likely to yield unique assignments. The procedure used for sequence specific assignment is described in Protocol 8. Protocol 8. Sequence specific assignment 1. Choose an amino acid type that occurs frequently in the sequence as the starting point. 2. From the adjacent residue list (Protocol 7) obtain the triplets of sequential residues (i-1,i,i+1) which have the chosen amino acid type at their centre (i). Compare the amino acid identifiers of these triplets with the known sequence of the protein and look for assignments for the triplet which are consistent with the sequence. If a triplet occurs only once in the sequence then a unique assignment can be made. 3. Once a triplet has been assigned look for spin systems adjacent to residues i-1 and i+1, that is, obtain from the adjacent residue list triplets with peaks i-1 and i+1 at their centre. Extend the segment in both directions until no further sequential connectivities are found. Check that the spin system identifiers of these residues are consistent with the protein sequence. 4. If more than one possible assignment exists for a particular triplet then the residues adjacent to i-1 and i+1 should be used. Compare the amino acid identifiers of the quintet of sequential residues (i-2,i- 1,i,i+1,i+2) with the known sequence of the protein and look for assignments which would be consistent. If a quintet occurs only once in the sequence then a unique assignment can be made. 5. Continue to extend segments at both ends (step 4) until a unique assignment is found for each residue selected in step Extend assigned segments in both directions until a break is reached (step 3) and check that the spin system identifiers are consistent with the protein sequence. 7. Choose a second amino acid type as the starting point and repeat steps When all the cross peaks with specific amino acid identifiers have been assigned then only fairly short segments in the sequence will remain unassigned. At this point assignments can be made unambiguously on the basis of the J, U and X classifications alone. By following the procedure outlined in Protocol 8 it should be possible to obtain sequence specific assignments for the majority of spin systems identified in stage 1. The assignments obtained should be entirely self consistent. If there are any discrepancies in the data then the assignments should be reviewed carefully for possible errors. Once all the spin systems have been assigned it is possible to go back and determine whether the breaks that occur between the sequential segments can be justified. 8

9 For example, do breaks occur at proline spin systems? If so, can the appropriate d αδ and d δn connectivities be identified now that the spin system is known to belong to proline? Do breaks in the sequential assignment occur because adjacent residues have overlapping H N resonances? This procedure for spin system identification and sequential assignment based on 1 H NMR alone was used in the 1980 s for the assignment of the spectra of several proteins containing more than 120 residues. The availability of 15 N or 15 N/ 13 C labelling has meant that 1 H assignment methods are now used less frequently. However, if labelled protein is not available then the 1 H assignment procedures described here can be applied, in favourable circumstances, to proteins of up to 15 kd. 3. Sequential assignment methodology for 15 N-labelled proteins Advances in protein expression methods have meant that uniform labelling of recombinant proteins with 15 N is now straightforward and relatively inexpensive. The assignment strategy for 15 N labelled proteins is very similar to that outlined above for unlabelled proteins. The advantage of 15 N labelling is that much improved resolution is achieved by spreading out the peaks in 1 H- 1 H TOCSY and NOESY spectra into a third dimension using the 15 N chemical shift of the backbone amide. The problems of overlap that make assignment of proteins of more than 10 kd difficult using 1 H methods are removed to a large extent using the 15 N-edited TOCSY and NOESY methods. In favourable circumstances proteins of up to ~20 kd can be assigned using this approach. 3.1 Optimising the 15 N- 1 H HSQC spectrum The 2D 15 N- 1 H HSQC spectrum, in the 15 N assignment strategy, is similar in importance to the 1 H- 1 H COSY spectrum, in the 1 H assignment strategy. If a good quality HSQC spectrum cannot be obtained for the protein of interest then the 15 N assignment approach, and the triple-resonance approaches described later, are unlikely to be successful and the recent TROSY-based methods should be applied. The HSQC spectrum of a 15 N-labelled protein will contain one-bond 1 H- 15 N connectivities for all backbone amides and for some chain nitrogens of Asn, Arg, Gln, His, Lys and Trp. Before collecting 3D data the acquisition parameters for the HSQC spectrum should be optimised in order to ensure good resolution in the 3D spectra; this procedure is described in Protocol 9. Protocol 9. Optimising the 1 H- 15 N HSQC spectrum. 1. The optimal 15 N sweep width and 15 N frequency will vary from protein to protein. The HSQC spectrum can be collected initially using a large sweep width in the indirect dimension (~50 ppm) centred at ~118 ppm. The HSQC spectrum may contains peaks that are shifted out of the main 15 N envelope by several ppm. It may be desirable to fold, or alias, these outlying peaks in order to decrease the 15 N sweep width. Identify the desired upper and lower frequencies in the 15 N dimension, these will determine the optimal sweep width. The 15 N frequency should be set to the average of the upper and lower frequencies selected. Collect the HSQC spectrum again with the optimised 15 N sweep width and carrier frequency. 2. The HSQC spectrum should contain one peak from the backbone amide of each residue with the exception of proline and the N-terminal residue. In addition, pairs of cross peaks, sharing a common 15 N shift, will be observed for each Asn and Gln side chain; these are found in the upfield region of both the 1 H and 15 N dimensions. A single peak will be observed for each Trp indole H ε1 in the downfield region of both the 1 H and 15 N dimensions. Depending on the ph and temperature of the sample peaks may be observed from Arg, His and Lys side chains. The chemical shifts of these nitrogens differ substantially from those of the backbone amides and these peaks will be folded in the HSQC spectrum. Count the number of observed peaks and compare with the number expected on the basis of the amino acid sequence. 3. On modern NMR spectrometers HSQC spectra can be collected using pulsed-field gradients and water flip-back methods. This eliminates the need for solvent presaturation and reduces the loss of H N peaks as a result of cross saturation. At ph values below 6-7 cross peaks should be observed from all backbone amides. At higher ph values the intrinsic H N exchange rates may become so fast that H N peaks are lost. 9

10 If a significant number of peaks are missing from the HSQC spectrum then try decreasing the ph or lowering the temperature and repeating the HSQC experiment. 4. Assign an arbitrary label to each cross peak observed in the HSQC spectrum. These labels will be used throughout the assignment process for bookkeeping purposes. Label the peaks from the most downfield 1 H chemical shift to the most upfield one; this is how data will be sorted in the 3D TOCSY-HSQC and NOESY-HSQC experiments D experiments for 15 N-labelled proteins. There are four 15 N-edited 3D experiments that can be useful for assignment. The most useful experiments are the TOCSY-HSQC and NOESY-HSQC; these should be collected initially. The other two experiments, HSQC-NOESY-HSQC and HNHA, may be useful but should only be collected after some analysis has been carried out using the first two data sets. The TOCSY-HSQC and the HNHA experiments provide through-bond spin system information. The NOESY-HSQC and HSQC-NOESY- HSQC experiments provide through-space NOE connectivities. The TOCSY-HSQC and NOESY-HSQC spectra are similar to their 2D 1 H- 1 H counterparts. These spectra contain a subset of the peaks in the 1 H spectra; only TOCSY or NOESY effects involving at least one H N are observed because the final component of the pulse sequences selects for 1 H bonded to 15 N. The 1 H- 1 H TOCSY or NOESY peaks are separated into 64 or 128 separate planes on the basis of the 15 N chemical shift of the backbone amide nitrogen. Thus, each 15 N plane in the 3D spectrum contains a small subset of the information from the 1 H- 1 H spectrum and overlap is reduced considerably. Much of the 3D matrix contains only noise and it is efficient to reduce the information in the 3D data set to a 2D strip plot as described in protocol 10. Protocol 10. Creating a 2D strip plot 1. For 15 N-edited TOCSY-HSQC and NOESY-HSQC spectra strips are extracted from 15 N planes. Each strip is centred about a H N chemical shift in F 3 and the strip covers the full 1 H sweep width in the indirect 1 H dimension, F 1. In order to make a 2D strip plot from a 3D matrix it is necessary to identify the H N (F 3 )- 15 N(F 2 ) positions at which the strips parallel to F 1 should be extracted. This can be done from peak positions in an HSQC spectrum collected prior to the 3D experiment or from peak positions in a 2D matrix created by projection of the 3D matrix along F The strips should be ordered according to the H N chemical shift; the most downfield H N is strip 1 and the most upfield H N is the last strip. The numbering of the strips corresponds to the arbitrary peak numbers assigned in the HSQC spectrum (Protocol 9). 3.3 Spin system information from 3D TOCSY-HSQC and 3D HNHA. The TOCSY-HSQC spectrum contains spin system information for each 15 N-H N pair. This information is identical to that contained in 1 H- 1 H TOCSY spectra but there will be less overlap as a result of the 15 N dimension. The rules for assigning spin system type on the basis of the TOCSY-HSQC spectrum are identical to those used in the analysis of 1 H- 1 H 2D TOCSY spectra described in section The amount of information contained in the 3D TOCSY-HSQC spectrum will depend on the length of the TOCSY mixing time, the linewidths of 1 H resonances, and the size of the H N -H α and H α -H β coupling constants. The TOCSY-HSQC spectrum will usually contain more spin system information for a β-sheet protein than for a helical one. Although the amount of spin system information will, in principle, increase as the TOCSY mixing time is increased, transverse relaxation will result in a decrease in signal at longer mixing times. On the basis of the rules outlined in Protocol 6 each 15 N-H N pair can be assigned to a more or less specific spin system category. For some proteins the spin system information that is obtained from 3D TOCSY-HSQC may not be sufficient to allow full sequential assignment. Spin system information that can be obtained from the 1 H- 1 H methods described earlier can be used to complement the information available from TOCSY-HSQC alone. The 3D HNHA spectrum contains H N -H N diagonal peaks and H N -H α cross peaks. The ratio of 10

11 these peak intensities provides quantitative information about the 3 J HNHα coupling constant. In addition, this data set can be used to identify, unambiguously, H α shifts for each residue. For example, comparison of HNHA and TOCSY-HSQC spectra may be useful for distinguishing Gly, which will give a pair of H α peaks in both spectra, from Ser or Thr, which give a single H α peak in the HNHA but may give 2 or 3 peaks in the ppm region of the TOCSY-HSQC spectrum due to the downfield shifted H β. 3.4 Sequential assignments from 3D NOESY-HSQC and HSQC-NOESY-HSQC The 3D NOESY-HSQC spectrum contains 1 H- 1 H NOEs involving at least one H N. Analysis of this spectrum for sequential assignment is based on the same rules as described in section 2.3. The NOE effects that can be identified most unambiguously from this spectrum are the H N -H N NOEs because they appear twice in the dataset. For example, an NOE between H N (i) and H N (j) will be observed in both the strip corresponding to H N (i) and that of H N (j). NOEs that involve a H N and a CH are only observed in the strip corresponding to the H N. The methods for analysing the NOESY-HSQC spectrum are outlined in Figure 5 and in Protocol 11. Protocol 11. Sequential assignment from 3D NOESY-HSQC 1. Identify intraresidue NOEs in the NOESY-HSQC strip plot by comparison with the TOCSY-HSQC strips. Label the H α for each residue and any H β or other side chain resonances that have been identified in the TOCSY spectrum. 2. Starting with the most downfield H N (i.e. the first strip) look in the region between 6 and 10 ppm in the indirect 1 H dimension for NOE cross peaks. For each cross peak observed look for a strip which has its diagonal peak at this 1 H frequency and a cross peak with a 1 H frequency corresponding to the original diagonal peak. These two symmetric strips represent a pair of H N s, which are correlated by a d NN NOE. If the cross peaks are strong then the pair of H N s are likely to represent sequential residues located in a helix or turn. If the cross peaks are weak then the NOE may be an interstrand contact in a β-sheet. The sequential connectivity can be confirmed by identifying additional sequential NOEs, d αn (i,i+1) and d βn (i,i+1). Place the two strips side by side and try to identify an NOE between the H N of one residue and the H α or H β of the other residue. Identification of such an NOE will confirm the assignment of the residues as neighbours in the sequence and will distinguish residue i and residue i+1. If appropriate d αn (i,i+1) or d βn (i,i+1) NOEs can not be seen for the two strips then the NOE may not be sequential. 3. Continue this procedure for all the NOEs observed in the region between 6 and 10 ppm. This will result in a list for each H N resonance of 0, 1 or 2 neighbouring H N s. Note that pairs of Asn and Gln side chain H N s also give rise to a strong H N -H N NOE cross peak. 4. If a strong cross peak is observed in the 6-10 ppm region but a reciprocal strip can not be identified then the cross peak involves an aromatic proton rather than a second H N. NOEs between H N s and aromatic side chains are often intraresidue or sequential so observation of this NOE may provide spin system information. 5. From the sequential d NN NOEs tabulated in 3 start to put together longer stretches of d NN connectivities. Breaks in these segments will occur at Pro, at the end of helical regions or because of the degeneracy of neighbouring H N shifts. However, because of the improved resolution compared to 2D 1 H methods longer segments of unambiguous connectivities should be seen. For each stretch of residues compare the spin system classification based on the TOCSY and other spin system experiments with the sequence of the protein (see Protocol 8). If the segment is long then even very limited spin system information will result in a unique assignment. 6. When all possible d NN NOEs have been identified the remaining assignments must be based on the strong sequential d αn NOEs that are expected in extended structure. Strips which did not show one or more strong d NN NOEs are likely to contain a strong interresidue d αn NOE. Because of the lack of symmetry in these NOEs unambiguous assignment may be more difficult. Sequential assignment based on d αn NOEs requires that strips are matched up according to H α shifts. A second strip plot in which strips are plotted in order of decreasing H α shifts can aid in this stage of analysis. 11

12 7. For each strip containing a strong interresidue d αn NOE look for a strip with an intraresidue d αn NOE at this chemical shift; there may be several such strips. Confirm that this is a sequential NOE by identifying the appropriate intra and interresidue d βn NOEs. In proteins with a high helical content and poor chemical shift dispersion in the H N region identification of sequential d NN NOEs may be difficult. The 3D HSQC-NOESY-HSQC spectrum may be useful for these proteins. In this experiment only d NN NOEs are observed. The indirect 1 H dimension of the NOESY-HSQC is replaced by a second 15 N dimension; the 15 N frequency of one residue is correlated with the 15 N and H N frequencies of a second residue. This experiment allows d NN NOEs to be observed between residues with degenerate H N chemical shifts as long as they have distinct 15 N shifts. This experiment is not useful for β-sheet proteins where very few H N -H N NOEs are observed. The assignment of the spectra of β-sheet proteins using 15 N-edited methods can be difficult because of ambiguities in the identification of d αn NOEs. The d NN NOEs that can be identified unambiguously often represent long-range contacts that may not be useful in the sequential assignment process. The main-chain directed (MCD) strategy, developed by Englander and Wand for analysis of 1 H NOE spectra (6), can be useful for analysis of 3D NOESY-HSQC spectra of β-sheet proteins. This strategy uses cyclic patterns involving residues on adjacent strands of the β-sheet. The two MCD patterns for the antiparallel β-sheet are designated as the outer and inner loops. The outer loop involves 2 TOCSY peaks (or intraresidue NOEs) and 3 interresidue NOEs: d αn (i,i), d αn (i+1,i+1), d αn (i,i+1), d αn (i+1,j) and d NN (i,j), where i and j are residues on opposite strands. This loop contains one interstrand d NN NOE that can be identified unambiguously. The inner loop involves 4 NOE effects: d αn (i+1,i+2), d αn (i+1,j), d αn (j- 1,j), and d αn (j-1,i+2); the d αα (i+1,j-1) NOE usually associated with this loop won't be observed in a 15 N- edited experiment. The inner and outer loops share a common NOE, d αn (i+1,j), and the H N of residue i+2, identified in the inner loop, is the starting point for the next outer loop with a d NN NOE to residue j-2. The MCD pattern for parallel β-sheet involves 2 TOCSY peaks (or intraresidue NOE's) and 4 interresidue NOEs: d αn (i,i), d αn (i+1,i+1), d αn (i,i+1), d αn (i+1,j+1), d αn (j,j+1) and d αn (j,i). Adjacent MCD loops in the parallel sheet share a common interstrand d αn NOE. The MCD pattern of the parallel sheet is less likely to give rise to unambiguous assignments because it does not involve a d NN NOE as a starting point. Nevertheless, if assignment using the sequential approach proves difficult the MCD approach should be considered. The 15 N-based sequential assignment method can be used to assign proteins up to ~20 kd in size but will not always be successful. In proteins with poor chemical shift dispersion the degeneracy of CH resonances will lead to ambiguities that may not be resolvable. In these circumstances double labelling with 15 N and 13 C may be required. 4. Triple-resonance assignment methods The triple-resonance assignment strategy is fundamentally different from the sequential assignment methodology described above. The latter method depends on through-space NOE effects for sequential connectivities because there is no resolved spin-spin coupling between 1 H resonances of adjacent residues. The triple-resonance methods, which require uniformly 15 N/ 13 C double-labelled protein, rely on spin-spin couplings to identify neighbouring residues. One bond couplings between 1 H, 15 N and 13 C vary in magnitude from ~5 to 140 Hz as shown in Figure 6. This methodology was first introduced in 1990 and was based on a series of four 3D triple resonance experiments and a 3D TOCSY- HMQC (7). Two of the triple resonance experiments were collected in H 2 O and two in D 2 O leading to differences in the measured C α and 15 N shifts due to H/D isotope effects. A large number of tripleresonance pulse sequences have been developed since 1990 and it is not possible to discuss all of these in this short chapter. In the protocols that follow several of the common approaches to assignment using triple-resonance experiments are outlined. The methods currently used involve detection of H N and all spectra are collected in H 2 O, thereby removing the problem of isotope shifts. Triple-resonance spectra 12

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