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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

Differences in the NH contributions to backbone entropy between the linear and circular SH3 constructs, on a per residue basis. (A) SH3lin-wt – SH3circ-Δ; (B) SH3lin-wt – SH3circ-GΔ; and (C) SH3lin-wt – SH3circ-wt.
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Figure 8: Differences in the NH contributions to backbone entropy between the linear and circular SH3 constructs, on a per residue basis. (A) SH3lin-wt – SH3circ-Δ; (B) SH3lin-wt – SH3circ-GΔ; and (C) SH3lin-wt – SH3circ-wt.

Mentions: The main structural differences between the linear and circular constructs are found in the circularization region and in the spatially proximal sites located in the β2 strand (Figure 7). These results are fully consistent with the observed chemical shift perturbations. For example, the resonance frequency for Ile161 HN is shifted upfield in SH3circ−GΔ, while in SH3circ−wt and SH3circ−Δ it is shifted downfield compared to SH3lin−wt (Figure 2). According to the crystal structure, the amide group of Ile161 forms a hydrogen bond with the carbonyl of residue 135. In the calculated structure of SH3circ−GΔ, the hydrogen bond is less populated (5 out of 20 structures), partially because of the somewhat greater distance between the β1 and β2 strands, and partially due to greater conformational flexibility of the Gly residue in the position 135 in this construct. This causes an upfield shift in HN of Ile161, because of the weaker deshielding effect of the carbonyl group. In the other circular construct, SH3circ−wt, the hydrogen bond is formed in all structures, and the associated stronger deshielding effect is responsible for the downfield shift in HN of Ile161 compared to SH3lin−wt where the hydrogen bond is present in 17 out of 20 structures. Increased backbone rigidity in SH3circ−wt (Figure 8C) could also contribute to a greater stability of this hydrogen bond.


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)

Differences in the NH contributions to backbone entropy between the linear and circular SH3 constructs, on a per residue basis. (A) SH3lin-wt – SH3circ-Δ; (B) SH3lin-wt – SH3circ-GΔ; and (C) SH3lin-wt – SH3circ-wt.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Differences in the NH contributions to backbone entropy between the linear and circular SH3 constructs, on a per residue basis. (A) SH3lin-wt – SH3circ-Δ; (B) SH3lin-wt – SH3circ-GΔ; and (C) SH3lin-wt – SH3circ-wt.
Mentions: The main structural differences between the linear and circular constructs are found in the circularization region and in the spatially proximal sites located in the β2 strand (Figure 7). These results are fully consistent with the observed chemical shift perturbations. For example, the resonance frequency for Ile161 HN is shifted upfield in SH3circ−GΔ, while in SH3circ−wt and SH3circ−Δ it is shifted downfield compared to SH3lin−wt (Figure 2). According to the crystal structure, the amide group of Ile161 forms a hydrogen bond with the carbonyl of residue 135. In the calculated structure of SH3circ−GΔ, the hydrogen bond is less populated (5 out of 20 structures), partially because of the somewhat greater distance between the β1 and β2 strands, and partially due to greater conformational flexibility of the Gly residue in the position 135 in this construct. This causes an upfield shift in HN of Ile161, because of the weaker deshielding effect of the carbonyl group. In the other circular construct, SH3circ−wt, the hydrogen bond is formed in all structures, and the associated stronger deshielding effect is responsible for the downfield shift in HN of Ile161 compared to SH3lin−wt where the hydrogen bond is present in 17 out of 20 structures. Increased backbone rigidity in SH3circ−wt (Figure 8C) could also contribute to a greater stability of this hydrogen bond.

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