Local and long-range stability in tandemly arrayed tetratricopeptide repeats

The tetratricopeptide repeat (TPR) is a 34-aa alpha-helical motif that occurs in tandem arrays in a variety of different proteins. In natural proteins, the number of TPR motifs ranges from 3 to 16 or more. These arrays function as molecular scaffolds and frequently mediate protein-protein interactions. We have shown that correctly folded TPR domain proteins, exhibiting the typical helix-turn-helix fold, can be designed by arraying tandem repeats of an idealized TPR consensus motif. To date, three designed proteins, CTPR1, CTPR2, and CTPR3 (consensus TPR number of repeats) have been characterized. Their high-resolution crystal structures show that the designed proteins indeed adopt the typical TPR fold, which is specified by the correct positioning of key residues. Here, we present a study of the thermodynamic properties and folding kinetics of this set of designed proteins. Chemical denaturation, monitored by CD and fluorescence, was used to assess the folding and global stability of each protein. NMR-detected amide proton exchange was used to investigate the stability of each construct at a residue-specific level. The results of these studies reveal a stable core, which defines the intrinsic stability of an individual TPR motif. The results also show the relationship between the number of tandem repeats and the overall stability and folding of the protei

Figure 6

<p>(a) Structural characterisation of the <i>per</i>-methylated FucPent₂Gal₃ oligosaccharide by high-energy MALDI-CID. Arabidopsis root AGP extracts were sequentially hydrolysed with α-arabinofuranosidase and endo-<i>β</i>-(1→6)-galactanase and the hydrolysis products were purified, <i>per</i>-methylated and analysed by MALDI-ToF-MS as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093291#pone-0093291-g004" target="_blank">Figure 4b</a>. The oligosaccharide with <i>m/z</i> 1175.3 as selected for MALDI-CID analysis and was identified as Xyl-(1→3)-<i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara-(1→3)-<i>β</i>-Gal<i>p</i>-(1→6)-<i>β</i>-Gal<i>p</i>-(1→6)-Gal<i>p</i>. Glycosidic cross-ring fragments were identified according to the Domon and Costello nomenclature (1988). (b) HPAEC-PAD monosaccharide analysis of neutral sugars for the HILIC purified root oligosaccharide. Arabidopsis root AGP extracts were sequentially hydrolysed with α-arabinofuranosidase and endo-<i>β</i>-(1→6)-galactanase and the hydrolysis products were reductively aminated with 2-AA and separated with HILIC. The FucPent₂Gal₃ oligosaccharide was collected from the HILIC column and hydrolysed by TFA. The sugar content was identified by HPAEC-PAD monosaccharide analysis. (b) NMR analysis of the FucPent₂Gal₃ hexasaccharide. (i) H-1 strip plots from 2D ¹H-¹H TOCSY (blue) and ROESY (red) spectra showing the NOE connectivity arising from the Xyl-(1→3)-<i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara-(1→3)-<i>β</i>-Gal<i>p</i> glycosidic linkages. (ii) 2D ¹H-¹H ROESY spectrum of the <i>α</i>-H1 region showing the Fuc H-1/Ara H-1 NOE arising from their close proximity due to the <i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara glycosidic linkage. (iii) 2D ¹³C HSQC and H2BC spectra showing the assignment of ¹H,¹³C HSQC peaks using H2BC, which exclusively reveals sequential connections over two covalent bonds (Nyberg et al., 2005). The H-1 chemical shift of the non-reducing-end Xyl is consistent with an <i>α</i> configuration; the ¹³C resonance positions of Fuc C-3, Ara C-2 and Gal C-3 are downfield shifted consistent with their involvement in glycosidic linkages. (NB: Fuc C-6 was aliased in the spectra; the actual resonance frequency c. 16 ppm is shown for clarity.).</p

Phenotypic analysis of Arabidopsis <i>fut</i> mutants compared to wild-type plants grown on solid medium.

<p>Wild-type, <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> seedlings were grown on MS medium for 7 days and then transferred to cellophane layered MS-agar medium plates without any supplements (a), with 100 mM NaCl (b) and with 100 mM mannitol (c) and grown for additional 7 days. (d) <i>fut4/fut6</i> double mutants are salt sensitive. The root growth response of <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> mutant plants to various NaCl concentrations was compared with those of wild-type. Data are presented as percentage relative to the growth of wild-type on MS medium. Bars represent mean ± SD (n = 3). A significant difference was identified between wild type and <i>fut4/fut6</i> mutant plants as indicated by <i>p</i>>0.05 in students <i>t-</i>test. (e) <i>fut4/fut6</i> double mutants are not sensitive to osmotic stress. The root growth response of <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> mutant plants to various mannitol concentrations was compared with those of wild-type. Data are presented as percentage relative to the growth of wild-type on MS medium. Bars represent mean ± SD (n = 3).</p

Capillary HILIC-MALDI-ToF-MS using stable isotope tagging of fucosylated oligosaccharides from Arabidopsis leaf AGP extracts of Columbia wild-type (Col-0; black line), <i>fut4</i> (red line), <i>fut6</i> (blue line) and <i>fut4/fut6</i> (magenta line) plants.

<p>Purified leaf AGP extracts were subjected to sequential digestion with α-arabinofuranosidase, exo-β-(1→3)-galactanase, β-glucuronidase and endo-β-(1→6)-galactanase. The oligosaccharide products were purified on a C₁₈ cartridge (elution with 5% acetic acid) and cation exchange resin (Dowex; elution with 5% acetic acid) and were reductively aminated with [¹²C₆]-aniline (wild-type oligosaccharides; black line) and [¹³C₆]-aniline (<i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i>; red, blue and magenta lines respectively). The labelled oligosaccharides were purified from the reductive amination reagents on a normal phase cartridge (Glyko clean S) and the purified glycans were separated by HILIC and analysed by MALDI-ToF-MS. Although all three fucosylated oligosaccharides were detected for wild-type and <i>fut6</i> samples, no corresponding glycans were detected from AGP leaf extracts from <i>fut4</i> and <i>fut4/fut6</i> plants. Background peaks are marked with an asterisk (*). On panel b the Col-0 trace (black line) partly coincides with, and therefore obscures, the <i>fut6</i> (blue line) trace.</p

EIC for the L-Fuc modified oligosaccharides from Arabidopsis root AGP extracts of wild-type (black line), <i>fut4</i> (red line), <i>fut6</i> (blue line) and <i>fut4/fut6</i> (magenta line) plants.

<p>Although all three fucosylated oligosaccharides were detected for wild-type, <i>fut4</i> and <i>fut6</i> samples, no FucAraGal₃ and FucAraGal₄ were detected from AGP root extracts from <i>fut4/fut6</i> plants and very small amount of FucPent₂Gal₃ oligosaccharide was detectable in root AGP extracts from <i>fut4/fut6</i> plants but its abundance relative to wild-type is significantly reduced.</p

HILIC purified oligosaccharide: HPAEC-PAD neutral monosaccharide analysis (% Mol).

<p>Values represent mean ± SD (n = 2).</p

RNA transcript levels of <i>FUT4</i> and <i>FUT6</i> genes by RT-PCR.

<p>RNA was isolated from roots 14 days after germination from homozygous <i>fut4</i>, <i>fut6</i>, <i>fut4/fut6</i> and wild-type seedlings and RT-PCR was performed using the primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093291#pone.0093291.s002" target="_blank">Table S1</a>. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. In lane 1 the <i>fut4</i> left (LP) and right (RP) primers were used. In lanes 2 and 3, the <i>fut6</i> and <i>gapdh</i> primers (LP and RP) respectively, were used.</p

Characterisation of oligosaccharides released by the sequential digestion with the AG-specific enzymes α-arabinofuranosidase and endo-β-(1→6)-galactanase from Arabidopsis root AGP extracts.

<p>(a) Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE). Oligosaccharide products from wild-type (Col-0), <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> were reductively aminated with 2-aminonaphthaline trisulfonic acid and separated by electrophoresis on acrylamide gels. An oligosaccharide ladder prepared from β-(1→6)-galactan was used as migration marker. The numbers indicate putatively fucosylated oligosaccharides with altered abundance in the wild-type and <i>fut</i> mutant samples. (b) MALDI-ToF-MS spectrum of <i>per</i>-methylated oligosaccharide products from wild-type plants. Peaks marked with an asterisk (*) were selected for high-energy MALDI-CID structural analysis. (c) Extracted ion chromatograms (EICs) for the fucosylated oligosaccharides originating from Arabidopsis leaf (blue lines) and root (red lines) AGP extracts hydrolysed sequentially by α-arabinofuranosidase, exo-β-(1→3)-galactanase, β-glucuronidase and endo-β-(1→6)-galactanase. Arabidopsis root AGP extracts contain the same three fucosylated oligosaccharides as leaves albeit in different relative abundances.</p

Examples for the determination of radial magnification errors.

<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (B—D) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p

Analysis of the rotor temperature.

<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p