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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 15N relaxation rates, R1, R2, and heteronuclear NOE, for the SH3 constructs studied here. Shown are data measured at 500 MHz (A–C) for SH3circ-Δ (red circles), SH3circ-GΔ (blue), and SH3lin-wt (green) and at 600 MHz (D–F) for SH3circ-GΔ (blue), SH3circ-wt (red squares), and SH3lin-wt (green).
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Figure 4: Comparison of the 15N relaxation rates, R1, R2, and heteronuclear NOE, for the SH3 constructs studied here. Shown are data measured at 500 MHz (A–C) for SH3circ-Δ (red circles), SH3circ-GΔ (blue), and SH3lin-wt (green) and at 600 MHz (D–F) for SH3circ-GΔ (blue), SH3circ-wt (red squares), and SH3lin-wt (green).

Mentions: In order to characterize the effect of circularization on the backbone dynamics of the SH3 domain, we measured 15N relaxation rates, R1 and R2, and the steady-state {1H}-15N NOE, as detailed in the Materials and Methods section. 48 to 50 well-resolved cross peaks from backbone amides were observed in the 1H-15N correlation maps. The relaxation data (Figure 4) are similar for most of the backbone amides in all SH3 constructs, suggesting that the subnanosecond backbone dynamics, by and large, are not significantly affected by the circularization. The rates of transverse relaxation show an interesting behavior. In all circular constructs, we observed elevated R2 values for the residues Glu166-Gln168 and Ala172 (in the β2/β3 loop and in the adjacent residues in the β2 and β3 strands), suggesting the presence of conformational exchange in this part of the backbone.


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 15N relaxation rates, R1, R2, and heteronuclear NOE, for the SH3 constructs studied here. Shown are data measured at 500 MHz (A–C) for SH3circ-Δ (red circles), SH3circ-GΔ (blue), and SH3lin-wt (green) and at 600 MHz (D–F) for SH3circ-GΔ (blue), SH3circ-wt (red squares), and SH3lin-wt (green).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Comparison of the 15N relaxation rates, R1, R2, and heteronuclear NOE, for the SH3 constructs studied here. Shown are data measured at 500 MHz (A–C) for SH3circ-Δ (red circles), SH3circ-GΔ (blue), and SH3lin-wt (green) and at 600 MHz (D–F) for SH3circ-GΔ (blue), SH3circ-wt (red squares), and SH3lin-wt (green).
Mentions: In order to characterize the effect of circularization on the backbone dynamics of the SH3 domain, we measured 15N relaxation rates, R1 and R2, and the steady-state {1H}-15N NOE, as detailed in the Materials and Methods section. 48 to 50 well-resolved cross peaks from backbone amides were observed in the 1H-15N correlation maps. The relaxation data (Figure 4) are similar for most of the backbone amides in all SH3 constructs, suggesting that the subnanosecond backbone dynamics, by and large, are not significantly affected by the circularization. The rates of transverse relaxation show an interesting behavior. In all circular constructs, we observed elevated R2 values for the residues Glu166-Gln168 and Ala172 (in the β2/β3 loop and in the adjacent residues in the β2 and β3 strands), suggesting the presence of conformational exchange in this part of the backbone.

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