Trigger Factor Slows Co-translational Folding through Kinetic Trapping while Sterically Protecting the Nascent Chain from Aberrant Cytosolic Interactions

The <i>E. coli</i> chaperone trigger factor (TF) interacts directly with nascent polypeptide chains as they emerge from the ribosome exit tunnel. Small protein domains can fold under the cradle created by TF, but the co-translational folding of larger proteins is slowed down by its presence. Because of the great experimental challenges in achieving high spatial and time resolution, it is not yet known whether or not TF alters the folding properties of small proteins and if the reduced rate of folding of larger proteins is the result of kinetic or thermodynamic effects. We show, by molecular simulations employing a coarse-grained model of a series of ribosome nascent-chain complexes, that TF does not alter significantly the co-translational folding process of a small protein G domain but delays that of a large β-galactosidase domain as a result of kinetic trapping of its unfolded ensemble. We demonstrate that this trapping occurs through a combination of three distinct mechanisms: a decrease in the rate of structural rearrangements within the nascent chain, an increase in the effective exit tunnel length due to folding outside the cradle, and entanglement of the nascent chain with TF. We present evidence that this TF-induced trapping represents a trade-off between promoting co-translational folding and sterically shielding the nascent chain from aberrant cytosolic interactions that could lead to its aggregation or degradation

Additional file 1: of Australian children living with rare diseases: experiences of diagnosis and perceived consequences of diagnostic delays

APSU Impact on Family Survey. (PDF 703 kb

Rapid Distinction of Intracellular and Extracellular Proteins Using NMR Diffusion Measurements

In-cell NMR spectroscopy offers a unique opportunity to begin to investigate the structures, dynamics, and interactions of molecules within their functional environments. An essential aspect of this technique is to define whether observed signals are attributable to intracellular species rather than to components of the extracellular medium. We report here the results of NMR measurements of the diffusion behavior of proteins expressed within bacterial cells, and find that these experiments provide a rapid and nondestructive probe of localization within cells and can be used to determine the size of the confining compartment. We show that diffusion can also be exploited as an editing method to eliminate extracellular species from high-resolution multidimensional spectra, and should be applicable to a wide range of problems. This approach is demonstrated here for a number of protein systems, using both ¹⁵N and ¹³C (methyl-TROSY) based acquisition

Comparison of secondary structure populations in isolated and intracellular αSyn.

<p>Secondary structure populations of αSyn calculated with the δ2D method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072286#pone.0072286-Camilloni1" target="_blank">[25]</a> using the backbone chemical shifts of (A) intracellular αSyn, (B) monomeric αSyn in bulk solution at the same pH, and (C) SDS micelle-bound αSyn at pH 7.4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072286#pone.0072286-Ulmer1" target="_blank">[12]</a> (D) Differences in secondary structure populations between intracellular and bulk solution measurements.</p

Analysis of backbone chemical shift changes and line broadening.

<p>(A) Overlay of ¹³C/¹⁵N slices through the HNCO spectrum of αSyn in bulk solution (green) and the deconvolved spectrum of αSyn expressed within cells (blue), showing resonances with ¹H chemical shifts between 8.45 and 8.55 ppm. (B–F) Backbone chemical shift changes observed for intracellular αSyn relative to the protein in bulk solution. (G) Relative HNCO intensities of intracellular αSyn compared to αSyn in bulk aqueous solution.</p

Clinical phenotypes associated with <i>GARS</i> variants in human.

<p>Clinical phenotypes associated with <i>GARS</i> variants in human.</p

Dipstick MRC enzyme data from cultured fibroblasts.

<p>Enzyme activity data are expressed as % residual activity relative to protein (% protein).</p

Multidimensional deconvolution of in-cell NMR spectra.

<p>HNCO spectra of αSyn (A) expressed within <i>E. coli</i> cells and (B) following deconvolution using the T92 reference peak indicated in (A). (C,D) ¹H/¹⁵N projections of (C) the original HNCO spectrum and (D) the spectrum following deconvolution.</p

Spectrophotometric MRC enzyme diagnostic data in skeletal muscle, liver and fibroblasts.

<p>Patient liver and muscle samples. Data are expressed as activity relative to protein and as % CS ratio, which represents % of the normal control mean value when expressed relative to Citrate Synthase. Bold characters indicate clinically significant abnormal values (H–high, L–low). Complex I (CI), NADH-coenzyme Q1 oxidoreductase; Complex II (CII), succinate-coenzyme Q1 oxidoreductase; Complex III (CIII), decylbenzylquinol-cytochrome c oxidoreductase; Complex IV, cytochrome c oxidase (CIV).</p

Compound heterozygous mutations in glycyl-tRNA synthetase (<i>GARS</i>) cause mitochondrial respiratory chain dysfunction - Fig 1

<p><b>A</b>) Sanger sequencing profile of <i>GARS</i> from the proband and parents showing c.803C>T; p.(Thr268Ile) variant is heterozygous in the proband and the father. <b>B)</b> Sanger sequencing profile of <i>GARS</i> from the proband and parents showing c.1234C>T; p.(Arg412Cys) variant is heterozygous in the proband and the mother. <b>C)</b> Evolutionary sequence conservations of the altered amino acid residues p.Thr268 and p. Arg412 are denoted in bold red in boxes.</p