Limits...
Contribution of protein phosphorylation to binding-induced folding of the SLBP-histone mRNA complex probed by phosphorus-31 NMR.

Thapar R - FEBS Open Bio (2014)

Bottom Line: Phosphorus-31 ((31)P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins.Here, I use (31)P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein).SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP-RNA complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Phosphorus-31 ((31)P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins. Here, I use (31)P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein). SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP-RNA complex. The data show that the (31)P chemical shift can be a good spectroscopic probe for phosphate-coupled folding and binding processes in intrinsically disordered proteins, particularly where the phosphate exhibits torsional strain and is involved in a network of hydrogen-bonding interactions.

No MeSH data available.


Related in: MedlinePlus

31P NMR spectra of the SLBP RBD–RNA complex collected at 25 °C at a spectrometer frequency of 202 MHz and at different pH values as indicated. A detailed pH titration is shown in Supplementary materials (Fig. S2).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215118&req=5

f0010: 31P NMR spectra of the SLBP RBD–RNA complex collected at 25 °C at a spectrometer frequency of 202 MHz and at different pH values as indicated. A detailed pH titration is shown in Supplementary materials (Fig. S2).

Mentions: In the absence of RNA, two 31P NMR resonances are observed for a single phosphate in the hSLBP RBD (Fig. 2) at basic pH (pH > 8.5) at 3.00 p.p.m and 4.19 p.p.m, both of which lie within the range of that expected for o-phosphothreonine (3–5 p.p.m) [15]. The linewidths for these resonances are broad, consistent with the hypothesis that this domain undergoes conformational exchange between multiple states as previously reported for both Drosophila[13] and human [15] SLBP RBDs. In the absence of RNA, unphosphorylated and phosphorylated hSLBP and dSLBP RBDs are intrinsically disordered [13,15] with a large hydrodynamic radius that is characteristic of a pre-molten globule state [13]. At pH 8.1, the upfield-shifted resonance has an apparent linewidth of 28.2 Hz while the downfield resonance has an apparent linewidth of 89.5 Hz. This broad resonance comprises a number of conformational sub-states that are only slightly different in chemical shift at pH 8.1, but become more apparent at pH 7.0 (Fig. 2). When RNA is titrated into the free protein, the phosphothreonine resonance undergoes a remarkable downfield-shift (Fig. 2) to a resonance position (∼20 p.p.m downfield of 85% H3PO4) that may be attributed to a deviation of the O–P–O σ-bond angle from that of a perfect tetrahedron (109°28′) to that observed in a five-membered cyclic phosphate ester (I) [24] (Fig. 3) or an increase in chemical-shift anisotropy due to the electronegativity of the phosphate due to next-nearest-neighbor ligands as seen in cation–phosphate interactions [25]. It is not the chemical shift expected for a free dianionic orthophosphate (II) (Fig. 3). At least two conformations are observed in the RNA bound complex as well (at 20.02 and 20.3 p.p.m) suggesting that it remains dynamic when bound to RNA in solution.


Contribution of protein phosphorylation to binding-induced folding of the SLBP-histone mRNA complex probed by phosphorus-31 NMR.

Thapar R - FEBS Open Bio (2014)

31P NMR spectra of the SLBP RBD–RNA complex collected at 25 °C at a spectrometer frequency of 202 MHz and at different pH values as indicated. A detailed pH titration is shown in Supplementary materials (Fig. S2).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215118&req=5

f0010: 31P NMR spectra of the SLBP RBD–RNA complex collected at 25 °C at a spectrometer frequency of 202 MHz and at different pH values as indicated. A detailed pH titration is shown in Supplementary materials (Fig. S2).
Mentions: In the absence of RNA, two 31P NMR resonances are observed for a single phosphate in the hSLBP RBD (Fig. 2) at basic pH (pH > 8.5) at 3.00 p.p.m and 4.19 p.p.m, both of which lie within the range of that expected for o-phosphothreonine (3–5 p.p.m) [15]. The linewidths for these resonances are broad, consistent with the hypothesis that this domain undergoes conformational exchange between multiple states as previously reported for both Drosophila[13] and human [15] SLBP RBDs. In the absence of RNA, unphosphorylated and phosphorylated hSLBP and dSLBP RBDs are intrinsically disordered [13,15] with a large hydrodynamic radius that is characteristic of a pre-molten globule state [13]. At pH 8.1, the upfield-shifted resonance has an apparent linewidth of 28.2 Hz while the downfield resonance has an apparent linewidth of 89.5 Hz. This broad resonance comprises a number of conformational sub-states that are only slightly different in chemical shift at pH 8.1, but become more apparent at pH 7.0 (Fig. 2). When RNA is titrated into the free protein, the phosphothreonine resonance undergoes a remarkable downfield-shift (Fig. 2) to a resonance position (∼20 p.p.m downfield of 85% H3PO4) that may be attributed to a deviation of the O–P–O σ-bond angle from that of a perfect tetrahedron (109°28′) to that observed in a five-membered cyclic phosphate ester (I) [24] (Fig. 3) or an increase in chemical-shift anisotropy due to the electronegativity of the phosphate due to next-nearest-neighbor ligands as seen in cation–phosphate interactions [25]. It is not the chemical shift expected for a free dianionic orthophosphate (II) (Fig. 3). At least two conformations are observed in the RNA bound complex as well (at 20.02 and 20.3 p.p.m) suggesting that it remains dynamic when bound to RNA in solution.

Bottom Line: Phosphorus-31 ((31)P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins.Here, I use (31)P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein).SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP-RNA complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.

ABSTRACT
Phosphorus-31 ((31)P) NMR can be used to characterize the structure and dynamics of phosphorylated proteins. Here, I use (31)P NMR to report on the chemical nature of a phosphothreonine that lies in the RNA binding domain of SLBP (stem-loop binding protein). SLBP is an intrinsically disordered protein and phosphorylation at this threonine promotes the assembly of the SLBP-RNA complex. The data show that the (31)P chemical shift can be a good spectroscopic probe for phosphate-coupled folding and binding processes in intrinsically disordered proteins, particularly where the phosphate exhibits torsional strain and is involved in a network of hydrogen-bonding interactions.

No MeSH data available.


Related in: MedlinePlus