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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

Comparison of the backbone dynamics in the circular and linear SH3 constructs. Left panels depict the squared order parameters derived from relaxation measurements at (A) 500 MHz and (B) 600 MHz. The coloring scheme is the same as in Figure 4. Right panels represent the conformational exchange contributions to R2 for (C) SH3circ-Δ at 500 MHz, (D) SH3circ-GΔ at 500 MHz; (E) SH3circ-GΔ at 600 MHz; (F) SH3circ-wt and 600 Mhz, and (G) SH3lin-wt at 500 MHz. (H) is a ribbon representation of the 3D structure of SH3circ-Δ: the ribbon width (proportional to 1-S2) represents the amplitudes of sub-nanosecond motions while the red coloring indicates the sites involved in conformational exchange. For comparison with the 500 MHz data, the Rex values shown in panels (E) and (F) were reduced by a factor of (1.2)2 which represents the expected field dependence (∝Bo2) of the Rex term. SH3circ-GΔ data at both magnetic fields are shown here to illustrate the reproducibility of the results. A similar agreement was observed between Rex terms measured in SH3lin-wt at 600 MHz (not shown) and those in (G). Horizontal bars on the top indicate the location of the secondary structure elements.
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Figure 5: Comparison of the backbone dynamics in the circular and linear SH3 constructs. Left panels depict the squared order parameters derived from relaxation measurements at (A) 500 MHz and (B) 600 MHz. The coloring scheme is the same as in Figure 4. Right panels represent the conformational exchange contributions to R2 for (C) SH3circ-Δ at 500 MHz, (D) SH3circ-GΔ at 500 MHz; (E) SH3circ-GΔ at 600 MHz; (F) SH3circ-wt and 600 Mhz, and (G) SH3lin-wt at 500 MHz. (H) is a ribbon representation of the 3D structure of SH3circ-Δ: the ribbon width (proportional to 1-S2) represents the amplitudes of sub-nanosecond motions while the red coloring indicates the sites involved in conformational exchange. For comparison with the 500 MHz data, the Rex values shown in panels (E) and (F) were reduced by a factor of (1.2)2 which represents the expected field dependence (∝Bo2) of the Rex term. SH3circ-GΔ data at both magnetic fields are shown here to illustrate the reproducibility of the results. A similar agreement was observed between Rex terms measured in SH3lin-wt at 600 MHz (not shown) and those in (G). Horizontal bars on the top indicate the location of the secondary structure elements.

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)

Comparison of the backbone dynamics in the circular and linear SH3 constructs. Left panels depict the squared order parameters derived from relaxation measurements at (A) 500 MHz and (B) 600 MHz. The coloring scheme is the same as in Figure 4. Right panels represent the conformational exchange contributions to R2 for (C) SH3circ-Δ at 500 MHz, (D) SH3circ-GΔ at 500 MHz; (E) SH3circ-GΔ at 600 MHz; (F) SH3circ-wt and 600 Mhz, and (G) SH3lin-wt at 500 MHz. (H) is a ribbon representation of the 3D structure of SH3circ-Δ: the ribbon width (proportional to 1-S2) represents the amplitudes of sub-nanosecond motions while the red coloring indicates the sites involved in conformational exchange. For comparison with the 500 MHz data, the Rex values shown in panels (E) and (F) were reduced by a factor of (1.2)2 which represents the expected field dependence (∝Bo2) of the Rex term. SH3circ-GΔ data at both magnetic fields are shown here to illustrate the reproducibility of the results. A similar agreement was observed between Rex terms measured in SH3lin-wt at 600 MHz (not shown) and those in (G). Horizontal bars on the top indicate the location of the secondary structure elements.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of the backbone dynamics in the circular and linear SH3 constructs. Left panels depict the squared order parameters derived from relaxation measurements at (A) 500 MHz and (B) 600 MHz. The coloring scheme is the same as in Figure 4. Right panels represent the conformational exchange contributions to R2 for (C) SH3circ-Δ at 500 MHz, (D) SH3circ-GΔ at 500 MHz; (E) SH3circ-GΔ at 600 MHz; (F) SH3circ-wt and 600 Mhz, and (G) SH3lin-wt at 500 MHz. (H) is a ribbon representation of the 3D structure of SH3circ-Δ: the ribbon width (proportional to 1-S2) represents the amplitudes of sub-nanosecond motions while the red coloring indicates the sites involved in conformational exchange. For comparison with the 500 MHz data, the Rex values shown in panels (E) and (F) were reduced by a factor of (1.2)2 which represents the expected field dependence (∝Bo2) of the Rex term. SH3circ-GΔ data at both magnetic fields are shown here to illustrate the reproducibility of the results. A similar agreement was observed between Rex terms measured in SH3lin-wt at 600 MHz (not shown) and those in (G). Horizontal bars on the top indicate the location of the secondary structure elements.
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