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Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Schumann FH, Varadan R, Tayakuniyil PP, Grossman JH, Camarero JA, Fushman D - Front Chem (2015)

Bottom Line: On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site.In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work.These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland College Park, MD, USA.

ABSTRACT
Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

No MeSH data available.


Related in: MedlinePlus

Model-independent verification of the conformational exchange broadening using η vs. R2' plot. Shown are data for SH3circ-GΔ measured at 600 MHz. The data points with significant shift to the right (triangles) from the linear dependence η vs. R2' (Fushman and Cowburn, 1998) correspond to those residues (indicated) involved in conformational exchange, in excellent agreement with the results of our model-free analysis of 15N relaxation data (cf. Figure 5E). The solid line corresponds to 15N CSA of −160 ppm and a 20° angle between the 15N CSA and 1H-15N dipolar tensors. A rough estimate of Rex values directly from the horizontal shift in the data is in good agreement with those from model-free analysis (Figure 5E): 0.8 s−1(Ile161), 1.6 s−1 (Asp163), 4.9 s−1 (Glu166), 1.7 s−1 (Glu167), 2.9 s−1 (Gln168), 0.9 s−1 (Trp170), 1.0 s−1 (Ala172), all numbers were divided by (1.2)2 to scale to 500 MHz.
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Figure 6: Model-independent verification of the conformational exchange broadening using η vs. R2' plot. Shown are data for SH3circ-GΔ measured at 600 MHz. The data points with significant shift to the right (triangles) from the linear dependence η vs. R2' (Fushman and Cowburn, 1998) correspond to those residues (indicated) involved in conformational exchange, in excellent agreement with the results of our model-free analysis of 15N relaxation data (cf. Figure 5E). The solid line corresponds to 15N CSA of −160 ppm and a 20° angle between the 15N CSA and 1H-15N dipolar tensors. A rough estimate of Rex values directly from the horizontal shift in the data is in good agreement with those from model-free analysis (Figure 5E): 0.8 s−1(Ile161), 1.6 s−1 (Asp163), 4.9 s−1 (Glu166), 1.7 s−1 (Glu167), 2.9 s−1 (Gln168), 0.9 s−1 (Trp170), 1.0 s−1 (Ala172), all numbers were divided by (1.2)2 to scale to 500 MHz.

Mentions: The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).


Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain.

Schumann FH, Varadan R, Tayakuniyil PP, Grossman JH, Camarero JA, Fushman D - Front Chem (2015)

Model-independent verification of the conformational exchange broadening using η vs. R2' plot. Shown are data for SH3circ-GΔ measured at 600 MHz. The data points with significant shift to the right (triangles) from the linear dependence η vs. R2' (Fushman and Cowburn, 1998) correspond to those residues (indicated) involved in conformational exchange, in excellent agreement with the results of our model-free analysis of 15N relaxation data (cf. Figure 5E). The solid line corresponds to 15N CSA of −160 ppm and a 20° angle between the 15N CSA and 1H-15N dipolar tensors. A rough estimate of Rex values directly from the horizontal shift in the data is in good agreement with those from model-free analysis (Figure 5E): 0.8 s−1(Ile161), 1.6 s−1 (Asp163), 4.9 s−1 (Glu166), 1.7 s−1 (Glu167), 2.9 s−1 (Gln168), 0.9 s−1 (Trp170), 1.0 s−1 (Ala172), all numbers were divided by (1.2)2 to scale to 500 MHz.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389572&req=5

Figure 6: Model-independent verification of the conformational exchange broadening using η vs. R2' plot. Shown are data for SH3circ-GΔ measured at 600 MHz. The data points with significant shift to the right (triangles) from the linear dependence η vs. R2' (Fushman and Cowburn, 1998) correspond to those residues (indicated) involved in conformational exchange, in excellent agreement with the results of our model-free analysis of 15N relaxation data (cf. Figure 5E). The solid line corresponds to 15N CSA of −160 ppm and a 20° angle between the 15N CSA and 1H-15N dipolar tensors. A rough estimate of Rex values directly from the horizontal shift in the data is in good agreement with those from model-free analysis (Figure 5E): 0.8 s−1(Ile161), 1.6 s−1 (Asp163), 4.9 s−1 (Glu166), 1.7 s−1 (Glu167), 2.9 s−1 (Gln168), 0.9 s−1 (Trp170), 1.0 s−1 (Ala172), all numbers were divided by (1.2)2 to scale to 500 MHz.
Mentions: The ensembles of 20 lowest-target-function structures (backbone) for the linear (A) and circular SH3 constructs: (B) SH3circ-Δ, (C) SH3circ-GΔ, and (D) SH3circ-wt. A cartoon representation of the 3D structure of the linear N-terminal SH3 domain of c-Crk is shown in (E). For the circular constructs the arrows indicate the location of the cyclization loop. Colored red are those residues exhibiting conformational exchange in the submillisecond time scale (cf. Figures 5, 6). The figure was prepared using MolMol (Koradi et al., 1996).

Bottom Line: On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site.In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work.These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland College Park, MD, USA.

ABSTRACT
Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

No MeSH data available.


Related in: MedlinePlus