Limits...
Amino acid selective unlabeling for sequence specific resonance assignments in proteins.

Krishnarjuna B, Jaipuria G, Thakur A, D'Silva P, Atreya HS - J. Biomol. NMR (2010)

Bottom Line: The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments.A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites.Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

View Article: PubMed Central - PubMed

Affiliation: NMR Research Centre, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective 'unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly (13)C/(15)N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(12)CO( i )-(15)N( i+1)}-filtered HSQC, which aids in linking the (1)H(N)/(15)N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to (2)H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

Show MeSH
Experimental (a) 2D difference spectrum and (b) 2D {12COi–15Ni+1}-filtered HSQC spectrum for the different selectively unlabeled samples of Ubiquitin. The difference spectrum was obtained by appropriate scaling and subtraction of 2D [15N, 1H] HSQC spectrum acquired for the selectively unlabeled sample from that of the reference sample. Amino acid pairs selectively unlabeled in each sample are indicated in each spectra. The assignments of peaks are indicated by the single letter code followed by the residue number. c A plot of IRunlab,i/IRref,i: IRunlab,i = Iiunlab/Icontrolunlab and IRref,i = Iiref/Icontrolref where i denotes the residue number, I denotes volume of the peak and ‘control’ denotes a residue which does not undergo any effect of unlabeling in both selectively unlabeled and the reference sample. In this case the control residue chosen was G75. All residues that undergo the desired unlabeling are indicated as green bars and correspond to the pairs of amino acids indicated in the respective spectra in (a) and (b). Those residues which undergo unlabeling due to misincorporation of 14N are shown in red. In the case of (R, N) sample complete unlabeling of Arg and Asn was obtained (i.e., IRunlab,i/IRref ~ 0) and hence these residues are indicated in the top panel with the single letter code. E24 and G53 were not assigned and hence are absent along with Pro
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3020294&req=5

Fig5: Experimental (a) 2D difference spectrum and (b) 2D {12COi–15Ni+1}-filtered HSQC spectrum for the different selectively unlabeled samples of Ubiquitin. The difference spectrum was obtained by appropriate scaling and subtraction of 2D [15N, 1H] HSQC spectrum acquired for the selectively unlabeled sample from that of the reference sample. Amino acid pairs selectively unlabeled in each sample are indicated in each spectra. The assignments of peaks are indicated by the single letter code followed by the residue number. c A plot of IRunlab,i/IRref,i: IRunlab,i = Iiunlab/Icontrolunlab and IRref,i = Iiref/Icontrolref where i denotes the residue number, I denotes volume of the peak and ‘control’ denotes a residue which does not undergo any effect of unlabeling in both selectively unlabeled and the reference sample. In this case the control residue chosen was G75. All residues that undergo the desired unlabeling are indicated as green bars and correspond to the pairs of amino acids indicated in the respective spectra in (a) and (b). Those residues which undergo unlabeling due to misincorporation of 14N are shown in red. In the case of (R, N) sample complete unlabeling of Arg and Asn was obtained (i.e., IRunlab,i/IRref ~ 0) and hence these residues are indicated in the top panel with the single letter code. E24 and G53 were not assigned and hence are absent along with Pro

Mentions: Figure 5a and b shows the 2D [15N–1H] HSQC-difference spectrum and 2D {12COi–15Ni+1}-filtered HSQC of the four selectively unlabeled samples, respectively. The efficacy of 2D {12COi–15Ni+1}-filtered HSQC to suppress peaks from residues having 13C/15N labeled N-terminal (sequential) neighbors was tested using the reference (uniformly 13C/15N) labeled sample of Ubiquitin. No peaks were observed in the spectrum (Figure S1 of Supporting Information) which is due to the fact that all residues in the reference sample have 13C/15N labeled N-terminal neighbors. Analysis of the intensities of peaks corresponding to the unlabeled amino acids (i.e., IRunlab/IRref) is shown in Fig. 5c. For calculating IRunlab and IRref, G75 was chosen as a control residue as it is unaffected by selective unlabeling in any of the samples. In all the four pairs of samples, complete peak yields were obtained for residues, i (i.e., the amino acid type being unlabeled) and i + 1. This implies that the extent of unlabeling of a desired amino acid type is always complete. Similar analysis for the 15N-labeled samples of the three other major selectively unlabeled amino acid types is provided in Supporting Information (Figure S2 of Supporting Information). Note that if two consecutive amino acids (i.e., both i and i + 1) are unlabeled, the experiment will not detect resonances corresponding to the i + 1 residue. This happens in the case of Q40–Q41, I2–I3, I30–Q31 and I61–Q62 in Ubiquitin.Fig. 5


Amino acid selective unlabeling for sequence specific resonance assignments in proteins.

Krishnarjuna B, Jaipuria G, Thakur A, D'Silva P, Atreya HS - J. Biomol. NMR (2010)

Experimental (a) 2D difference spectrum and (b) 2D {12COi–15Ni+1}-filtered HSQC spectrum for the different selectively unlabeled samples of Ubiquitin. The difference spectrum was obtained by appropriate scaling and subtraction of 2D [15N, 1H] HSQC spectrum acquired for the selectively unlabeled sample from that of the reference sample. Amino acid pairs selectively unlabeled in each sample are indicated in each spectra. The assignments of peaks are indicated by the single letter code followed by the residue number. c A plot of IRunlab,i/IRref,i: IRunlab,i = Iiunlab/Icontrolunlab and IRref,i = Iiref/Icontrolref where i denotes the residue number, I denotes volume of the peak and ‘control’ denotes a residue which does not undergo any effect of unlabeling in both selectively unlabeled and the reference sample. In this case the control residue chosen was G75. All residues that undergo the desired unlabeling are indicated as green bars and correspond to the pairs of amino acids indicated in the respective spectra in (a) and (b). Those residues which undergo unlabeling due to misincorporation of 14N are shown in red. In the case of (R, N) sample complete unlabeling of Arg and Asn was obtained (i.e., IRunlab,i/IRref ~ 0) and hence these residues are indicated in the top panel with the single letter code. E24 and G53 were not assigned and hence are absent along with Pro
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020294&req=5

Fig5: Experimental (a) 2D difference spectrum and (b) 2D {12COi–15Ni+1}-filtered HSQC spectrum for the different selectively unlabeled samples of Ubiquitin. The difference spectrum was obtained by appropriate scaling and subtraction of 2D [15N, 1H] HSQC spectrum acquired for the selectively unlabeled sample from that of the reference sample. Amino acid pairs selectively unlabeled in each sample are indicated in each spectra. The assignments of peaks are indicated by the single letter code followed by the residue number. c A plot of IRunlab,i/IRref,i: IRunlab,i = Iiunlab/Icontrolunlab and IRref,i = Iiref/Icontrolref where i denotes the residue number, I denotes volume of the peak and ‘control’ denotes a residue which does not undergo any effect of unlabeling in both selectively unlabeled and the reference sample. In this case the control residue chosen was G75. All residues that undergo the desired unlabeling are indicated as green bars and correspond to the pairs of amino acids indicated in the respective spectra in (a) and (b). Those residues which undergo unlabeling due to misincorporation of 14N are shown in red. In the case of (R, N) sample complete unlabeling of Arg and Asn was obtained (i.e., IRunlab,i/IRref ~ 0) and hence these residues are indicated in the top panel with the single letter code. E24 and G53 were not assigned and hence are absent along with Pro
Mentions: Figure 5a and b shows the 2D [15N–1H] HSQC-difference spectrum and 2D {12COi–15Ni+1}-filtered HSQC of the four selectively unlabeled samples, respectively. The efficacy of 2D {12COi–15Ni+1}-filtered HSQC to suppress peaks from residues having 13C/15N labeled N-terminal (sequential) neighbors was tested using the reference (uniformly 13C/15N) labeled sample of Ubiquitin. No peaks were observed in the spectrum (Figure S1 of Supporting Information) which is due to the fact that all residues in the reference sample have 13C/15N labeled N-terminal neighbors. Analysis of the intensities of peaks corresponding to the unlabeled amino acids (i.e., IRunlab/IRref) is shown in Fig. 5c. For calculating IRunlab and IRref, G75 was chosen as a control residue as it is unaffected by selective unlabeling in any of the samples. In all the four pairs of samples, complete peak yields were obtained for residues, i (i.e., the amino acid type being unlabeled) and i + 1. This implies that the extent of unlabeling of a desired amino acid type is always complete. Similar analysis for the 15N-labeled samples of the three other major selectively unlabeled amino acid types is provided in Supporting Information (Figure S2 of Supporting Information). Note that if two consecutive amino acids (i.e., both i and i + 1) are unlabeled, the experiment will not detect resonances corresponding to the i + 1 residue. This happens in the case of Q40–Q41, I2–I3, I30–Q31 and I61–Q62 in Ubiquitin.Fig. 5

Bottom Line: The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments.A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites.Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

View Article: PubMed Central - PubMed

Affiliation: NMR Research Centre, Indian Institute of Science, Bangalore 560012, India.

ABSTRACT
Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective 'unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly (13)C/(15)N labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(12)CO( i )-(15)N( i+1)}-filtered HSQC, which aids in linking the (1)H(N)/(15)N resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to (2)H labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of (14)N at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

Show MeSH