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A study of the ultrastructure of fragile-X-related proteins.

Sjekloća L, Konarev PV, Eccleston J, Taylor IA, Svergun DI, Pastore A - Biochem. J. (2009)

Bottom Line: In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region.We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape.The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

ABSTRACT
Fragile-X-related proteins form a family implicated in RNA metabolism. Their sequence is composed of conserved N-terminal and central regions which contain Tudor and KH domains and of a divergent C-terminus with motifs rich in arginine and glycine residues. The most widely studied member of the family is probably FMRP (fragile X mental retardation protein), since absence or mutation of this protein in humans causes fragile X syndrome, the most common cause of inherited mental retardation. Understanding the structural properties of FMRP is essential for correlating it with its functions. The structures of isolated domains of FMRP have been reported, but nothing is yet known with regard to the spatial arrangement of the different modules, partly because of difficulties in producing both the full-length protein and its multidomain fragments in quantities, purities and monodispersity amenable for structural studies. In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region. We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape. The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

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Related in: MedlinePlus

Spectroscopic studies of FXR proteins(A) Far-UV CD spectra in molar ellipticity of the Nt-KH1 of FMRP (black continuous line), FXR1P (grey broken line) and FXR1P Nt-KH2 (grey continuous line). The measurements were carried out at 20 °C using 5 μM concentrations. (B) NMR HSQC spectra of Nt-KH1 from FXR1P (top panel) and FMRP (bottom panel). The spectra were recorded at 800 MHz and 25 °C, using concentrations of 60 μM (FXR1P) and 230 μM (FMRP).
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Figure 2: Spectroscopic studies of FXR proteins(A) Far-UV CD spectra in molar ellipticity of the Nt-KH1 of FMRP (black continuous line), FXR1P (grey broken line) and FXR1P Nt-KH2 (grey continuous line). The measurements were carried out at 20 °C using 5 μM concentrations. (B) NMR HSQC spectra of Nt-KH1 from FXR1P (top panel) and FMRP (bottom panel). The spectra were recorded at 800 MHz and 25 °C, using concentrations of 60 μM (FXR1P) and 230 μM (FMRP).

Mentions: The three well-behaving fragments were characterized by CD and NMR techniques to ensure that they were correctly folded. The far-UV CD spectra of FXR1 Nt-KH2 and Nt-KH1 and of FMRP Nt-KH1 are typical of well-folded proteins with features of a mixed αβ fold, as we expect from what we know about the structures of the Tudor and KH domains which must contribute to the signal (Figure 2A). The secondary structure content as estimated from the spectra was, as expected, very similar for the two Nt-KH1 constructs (approx. 21% α, 27% β, 20% turn and 32% random) and increased for FXR1P Nt-KH2 (28% α, 22% β, 20% turn and 30% random). This increase was roughly consistent with the increment expected by assuming the secondary structure content observed in KH1KH2Δ.


A study of the ultrastructure of fragile-X-related proteins.

Sjekloća L, Konarev PV, Eccleston J, Taylor IA, Svergun DI, Pastore A - Biochem. J. (2009)

Spectroscopic studies of FXR proteins(A) Far-UV CD spectra in molar ellipticity of the Nt-KH1 of FMRP (black continuous line), FXR1P (grey broken line) and FXR1P Nt-KH2 (grey continuous line). The measurements were carried out at 20 °C using 5 μM concentrations. (B) NMR HSQC spectra of Nt-KH1 from FXR1P (top panel) and FMRP (bottom panel). The spectra were recorded at 800 MHz and 25 °C, using concentrations of 60 μM (FXR1P) and 230 μM (FMRP).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2662490&req=5

Figure 2: Spectroscopic studies of FXR proteins(A) Far-UV CD spectra in molar ellipticity of the Nt-KH1 of FMRP (black continuous line), FXR1P (grey broken line) and FXR1P Nt-KH2 (grey continuous line). The measurements were carried out at 20 °C using 5 μM concentrations. (B) NMR HSQC spectra of Nt-KH1 from FXR1P (top panel) and FMRP (bottom panel). The spectra were recorded at 800 MHz and 25 °C, using concentrations of 60 μM (FXR1P) and 230 μM (FMRP).
Mentions: The three well-behaving fragments were characterized by CD and NMR techniques to ensure that they were correctly folded. The far-UV CD spectra of FXR1 Nt-KH2 and Nt-KH1 and of FMRP Nt-KH1 are typical of well-folded proteins with features of a mixed αβ fold, as we expect from what we know about the structures of the Tudor and KH domains which must contribute to the signal (Figure 2A). The secondary structure content as estimated from the spectra was, as expected, very similar for the two Nt-KH1 constructs (approx. 21% α, 27% β, 20% turn and 32% random) and increased for FXR1P Nt-KH2 (28% α, 22% β, 20% turn and 30% random). This increase was roughly consistent with the increment expected by assuming the secondary structure content observed in KH1KH2Δ.

Bottom Line: In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region.We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape.The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

View Article: PubMed Central - PubMed

Affiliation: National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.

ABSTRACT
Fragile-X-related proteins form a family implicated in RNA metabolism. Their sequence is composed of conserved N-terminal and central regions which contain Tudor and KH domains and of a divergent C-terminus with motifs rich in arginine and glycine residues. The most widely studied member of the family is probably FMRP (fragile X mental retardation protein), since absence or mutation of this protein in humans causes fragile X syndrome, the most common cause of inherited mental retardation. Understanding the structural properties of FMRP is essential for correlating it with its functions. The structures of isolated domains of FMRP have been reported, but nothing is yet known with regard to the spatial arrangement of the different modules, partly because of difficulties in producing both the full-length protein and its multidomain fragments in quantities, purities and monodispersity amenable for structural studies. In the present study, we describe how we have produced overlapping recombinant fragments of human FMRP and its paralogues which encompass the evolutionary conserved region. We have studied their behaviour in solution by complementary biochemical and biophysical techniques, identified the regions which promote self-association and determined their overall three-dimensional shape. The present study paves the way to further studies and rationalizes the existing knowledge on the self-association properties of these proteins.

Show MeSH
Related in: MedlinePlus