Limits...
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

Chemical shift perturbations for the circular constructs vs. linear SH3 domain. The left, middle, and right columns represent the difference, δ (linear)–δ (circular), between chemical shift positions in the linear and circular proteins, for 15N, 1HN, and 1Hα, respectively. The rows, from top to bottom, correspond to SH3circ-Δ, SH3circ-GΔ, and SH3circ-wt. In the case of Gly, shown is the largest of the chemical shift perturbations for the two α-protons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4389572&req=5

Figure 2: Chemical shift perturbations for the circular constructs vs. linear SH3 domain. The left, middle, and right columns represent the difference, δ (linear)–δ (circular), between chemical shift positions in the linear and circular proteins, for 15N, 1HN, and 1Hα, respectively. The rows, from top to bottom, correspond to SH3circ-Δ, SH3circ-GΔ, and SH3circ-wt. In the case of Gly, shown is the largest of the chemical shift perturbations for the two α-protons.

Mentions: Chemical shift is a sensitive indicator of changes in the electron environment (hence in local structure) of a nucleus under observation. The comparison of 15N, 1HN, and 1Hα chemical shifts in the circular constructs vs. SH3lin−wt (Figure 2) shows, as expected, some chemical shift differences for the residues in and immediately adjacent to the cyclization region. Tyr136 and Tyr190 are of particular interest here: involved in hydrogen bonding between the β5- and the β1-strands there residues are located at the edges of the strands, bordering the newly formed cyclization loop (Figure 3). A comparison of the magnitudes and directions of the shifts in 15N and 1HN resonance frequencies for Tyr190 in all the SH3 constructs revealed an increase in the deshielding effect from SH3circ−wt to SH3lin−wt to SH3circ−GΔ and then to SH3circ−Δ suggesting an increase in the stability of the hydrogen bond between the amide of Tyr190 and carbonyl of Tyr136 in these constructs. Even stronger chemical shift changes are observed in Tyr136; however, their interpretation is less straightforward, because of the additional effect of the change in the identity of the neighboring (N-terminal) residue. In addition, strong perturbations are observed in Leu159 -Arg162 (strand β2). These sites are located in close spatial proximity to the N-terminus and make several hydrogen bonds with the β1-strand, most notable between the amide of Lys161 and the carbonyl oxygen of residue 135 (Figure 3). The observed perturbations likely reflect some local structural rearrangements upon circularization. Except for these two regions, the overall similarity of the observed chemical shifts in the rest of the sequence indicates that the overall fold of the SH3 domain is preserved upon circularization.


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)

Chemical shift perturbations for the circular constructs vs. linear SH3 domain. The left, middle, and right columns represent the difference, δ (linear)–δ (circular), between chemical shift positions in the linear and circular proteins, for 15N, 1HN, and 1Hα, respectively. The rows, from top to bottom, correspond to SH3circ-Δ, SH3circ-GΔ, and SH3circ-wt. In the case of Gly, shown is the largest of the chemical shift perturbations for the two α-protons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Chemical shift perturbations for the circular constructs vs. linear SH3 domain. The left, middle, and right columns represent the difference, δ (linear)–δ (circular), between chemical shift positions in the linear and circular proteins, for 15N, 1HN, and 1Hα, respectively. The rows, from top to bottom, correspond to SH3circ-Δ, SH3circ-GΔ, and SH3circ-wt. In the case of Gly, shown is the largest of the chemical shift perturbations for the two α-protons.
Mentions: Chemical shift is a sensitive indicator of changes in the electron environment (hence in local structure) of a nucleus under observation. The comparison of 15N, 1HN, and 1Hα chemical shifts in the circular constructs vs. SH3lin−wt (Figure 2) shows, as expected, some chemical shift differences for the residues in and immediately adjacent to the cyclization region. Tyr136 and Tyr190 are of particular interest here: involved in hydrogen bonding between the β5- and the β1-strands there residues are located at the edges of the strands, bordering the newly formed cyclization loop (Figure 3). A comparison of the magnitudes and directions of the shifts in 15N and 1HN resonance frequencies for Tyr190 in all the SH3 constructs revealed an increase in the deshielding effect from SH3circ−wt to SH3lin−wt to SH3circ−GΔ and then to SH3circ−Δ suggesting an increase in the stability of the hydrogen bond between the amide of Tyr190 and carbonyl of Tyr136 in these constructs. Even stronger chemical shift changes are observed in Tyr136; however, their interpretation is less straightforward, because of the additional effect of the change in the identity of the neighboring (N-terminal) residue. In addition, strong perturbations are observed in Leu159 -Arg162 (strand β2). These sites are located in close spatial proximity to the N-terminus and make several hydrogen bonds with the β1-strand, most notable between the amide of Lys161 and the carbonyl oxygen of residue 135 (Figure 3). The observed perturbations likely reflect some local structural rearrangements upon circularization. Except for these two regions, the overall similarity of the observed chemical shifts in the rest of the sequence indicates that the overall fold of the SH3 domain is preserved upon circularization.

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