Protocols for the Sequential Solid-State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET-s(1-227) (original) (raw)

Abstract

. Extent of solid-state NMR backbone assignments displayed on the protein primary sequence. The secondary structure (residue 11-224) elements are given on top. Sequentially assigned residues are given in black. The GS prior to the initial M residue is not part of the protein primary sequence but remainders of the cleaved His-tag from protein purification.

Figures (8)

Figure 1. A) One-dimensional 'H NMR spectrum showing the broad resonance from crys- tal water and a minor, sharp line from supernatant water." The resonance at 3.75 ppm comes from the crystallization agent PEG 4000. B) 1D '3C-CP-MAS spectrum, 64 scans. C) INEPT spectrum revealing highly dynamic residues. No backbone resonances are de- tected, only flexible side chains. The two correlations marked with an asterisk do not cor- respond to random-coil chemical shifts and are not assigned. The spectra were recorded at 850 MHz, 18 kHz MAS (A and B) and 600 MHz, 13 kHz (C).

Figure 1. A) One-dimensional 'H NMR spectrum showing the broad resonance from crys- tal water and a minor, sharp line from supernatant water." The resonance at 3.75 ppm comes from the crystallization agent PEG 4000. B) 1D '3C-CP-MAS spectrum, 64 scans. C) INEPT spectrum revealing highly dynamic residues. No backbone resonances are de- tected, only flexible side chains. The two correlations marked with an asterisk do not cor- respond to random-coil chemical shifts and are not assigned. The spectra were recorded at 850 MHz, 18 kHz MAS (A and B) and 600 MHz, 13 kHz (C).

Figure 2. Two-dimensional spectra for initial sample characterization of crys- talline HET-s(1-227) recorded at 850 MHz 'H frequency, 18 kHz MAS. A) Close-up of the aliphatic region of a '*C—'?C DARR/MIRROR spectrum with 100 ms mixing time revealing intraresidual correlations of the C* spins. B) NCA spectrum revealing intraresidue (and very few interresidue) NCa cor- relations with the C* spins. The peak positions marked by crosses are taken, in both spectra, from the final chemical-shift assignment listed in Table S2. Some isolated peaks with a unique assignment are labeled.

Figure 2. Two-dimensional spectra for initial sample characterization of crys- talline HET-s(1-227) recorded at 850 MHz 'H frequency, 18 kHz MAS. A) Close-up of the aliphatic region of a '*C—'?C DARR/MIRROR spectrum with 100 ms mixing time revealing intraresidual correlations of the C* spins. B) NCA spectrum revealing intraresidue (and very few interresidue) NCa cor- relations with the C* spins. The peak positions marked by crosses are taken, in both spectra, from the final chemical-shift assignment listed in Table S2. Some isolated peaks with a unique assignment are labeled.

Figure 3. Statistics of the median distance (d) in ppm between nearest- neighbor peaks in generic two-dimensional spectra as a function of the size (number of residues) of the protein. Resonance frequencies were taken from assignments deposited in the BMRB.“ Only nonparamagnetic proteins with extended stretches with a complete assignment (N", C“, C® and C’) were taken into account. Amongst the different bonded spin pairs in the back- bone, correlations involving C’ clearly have the lowest spectral dispersion. The N,C*_, spin pair has the best dispersion followed by NC“, and C°C?, which are about equally dispersed. We can use these trends to judge the dispersion in the different 3D spectra. The NCACO, NCOCA and CANCO ex- periments rely on NC*, C°C’ and C’_,N; for dispersion whereas the set of ex- periments without C’ dimension, NCACB, N(CO)CACB, and CAN(CO)CA, rely on NC*, C“C®, and C%,_,N;. The second set of experiments is therefore expect- ed to give spectra with a significantly higher spectral resolution. This com- parison assumes that '°N and "°C linewidth are roughly equal in ppm which is the case in our sample.

Figure 3. Statistics of the median distance (d) in ppm between nearest- neighbor peaks in generic two-dimensional spectra as a function of the size (number of residues) of the protein. Resonance frequencies were taken from assignments deposited in the BMRB.“ Only nonparamagnetic proteins with extended stretches with a complete assignment (N", C“, C® and C’) were taken into account. Amongst the different bonded spin pairs in the back- bone, correlations involving C’ clearly have the lowest spectral dispersion. The N,C*_, spin pair has the best dispersion followed by NC“, and C°C?, which are about equally dispersed. We can use these trends to judge the dispersion in the different 3D spectra. The NCACO, NCOCA and CANCO ex- periments rely on NC*, C°C’ and C’_,N; for dispersion whereas the set of ex- periments without C’ dimension, NCACB, N(CO)CACB, and CAN(CO)CA, rely on NC*, C“C®, and C%,_,N;. The second set of experiments is therefore expect- ed to give spectra with a significantly higher spectral resolution. This com- parison assumes that '°N and "°C linewidth are roughly equal in ppm which is the case in our sample.

Figure 4. A) Schematic overview of the three-dimensional correlation experi- ments used for the sequential backbone resonance assignment in this study. Only the desired coherence transfers are shown. Minor pathways into the side chains, which in fact can be beneficial for assignment, are omitted for clarity. The transfer steps needed to walk sequentially from residue (i) to resi- due (i+1) are highlighted in bold lines. Indirect or direct acquisition is done on the spins in circles. The experiments can be classified into two catego- ries: those containing a carbonyl-resolved dimension and those with an ad- ditional transfer step and no evolution period on the carbonyl spins. B) Scheme of 3D experiments for side-chain resonances: N(CA)CBCX, NCACX, and CCC. CX is an arbitrary side-chain carbon spin. C) Assignment strategy by using the NCACB, N(CO)CACB, and CAN(CO)CA experiments. For a se- quential walk, two known frequencies (highlighted in yellow) are always used to connect to the next spin (highlighted in grey).

Figure 4. A) Schematic overview of the three-dimensional correlation experi- ments used for the sequential backbone resonance assignment in this study. Only the desired coherence transfers are shown. Minor pathways into the side chains, which in fact can be beneficial for assignment, are omitted for clarity. The transfer steps needed to walk sequentially from residue (i) to resi- due (i+1) are highlighted in bold lines. Indirect or direct acquisition is done on the spins in circles. The experiments can be classified into two catego- ries: those containing a carbonyl-resolved dimension and those with an ad- ditional transfer step and no evolution period on the carbonyl spins. B) Scheme of 3D experiments for side-chain resonances: N(CA)CBCX, NCACX, and CCC. CX is an arbitrary side-chain carbon spin. C) Assignment strategy by using the NCACB, N(CO)CACB, and CAN(CO)CA experiments. For a se- quential walk, two known frequencies (highlighted in yellow) are always used to connect to the next spin (highlighted in grey).

Figure 6. Representative planes from the three 3D spectra used for the back- bone assignment. All peaks correspond to sequential correlations picked in the spectra; the assignment is given where space permits. As two represen- tative amino acid neighbors, the correlations used to establish sequential contact from V22 to D23 are highlighted in red. A) 5,-63-Plane of the NCACB spectrum at 6,=117.8 ppm. The spin system of V22 is highlighted. B) 6,—63 plane of the N(CO)CACB spectrum at 6,=117.1 ppm. The connection of the V22 C"CP resonance pair to the N-resonance of D23 is highlighted. C) 8,—83 Plane of the CAN(CO)CA spectrum at 6,=117.1 ppm. The connection V22 CA to the NC*-resonance pair of D23 is highlighted.

Figure 6. Representative planes from the three 3D spectra used for the back- bone assignment. All peaks correspond to sequential correlations picked in the spectra; the assignment is given where space permits. As two represen- tative amino acid neighbors, the correlations used to establish sequential contact from V22 to D23 are highlighted in red. A) 5,-63-Plane of the NCACB spectrum at 6,=117.8 ppm. The spin system of V22 is highlighted. B) 6,—63 plane of the N(CO)CACB spectrum at 6,=117.1 ppm. The connection of the V22 C"CP resonance pair to the N-resonance of D23 is highlighted. C) 8,—83 Plane of the CAN(CO)CA spectrum at 6,=117.1 ppm. The connection V22 CA to the NC*-resonance pair of D23 is highlighted.

Figure 7. Sequential walk along the protein backbone by using NCACB, N(CO)CACB, and CAN(CO)CA experiments in the direction from the N to C terminus by following the scheme displayed in Figure 4A. For each strip, the previously unknown resonance (highlighted in gray in Figure 4C) is displayed in the verti- cal dimension. The potential ambiguity of an assignment can be judged from the number of peaks on the dashed line. An alternative representation of the same data is given in Figure $3.

Figure 7. Sequential walk along the protein backbone by using NCACB, N(CO)CACB, and CAN(CO)CA experiments in the direction from the N to C terminus by following the scheme displayed in Figure 4A. For each strip, the previously unknown resonance (highlighted in gray in Figure 4C) is displayed in the verti- cal dimension. The potential ambiguity of an assignment can be judged from the number of peaks on the dashed line. An alternative representation of the same data is given in Figure $3.

Figure 8. A) Representative 5,—6, plane from the N(CA)CBCX spectrum at 5, = 117.8 ppm. The peak corresponding to V22 is highlighted in red. B) 5,—53 plane from the CCC spec- trum at 6,=66.8 ppm. The spin system of V22 is highlighted in red. C) Statistics about side-chain assignments according to amino acid type. For each amino acid, the percentage of assigned side-chain carbon atoms (all except C*, CC’) for all assigned residues is given. More detailed statistics itemized by amino acid and atom type are given in Table S2.

Figure 8. A) Representative 5,—6, plane from the N(CA)CBCX spectrum at 5, = 117.8 ppm. The peak corresponding to V22 is highlighted in red. B) 5,—53 plane from the CCC spec- trum at 6,=66.8 ppm. The spin system of V22 is highlighted in red. C) Statistics about side-chain assignments according to amino acid type. For each amino acid, the percentage of assigned side-chain carbon atoms (all except C*, CC’) for all assigned residues is given. More detailed statistics itemized by amino acid and atom type are given in Table S2.

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