Biology subjects

Christodoulou, J.

Publications and source records attributed to Christodoulou, J..

23 records · Page 2Linked to original sources

bioRxiv2019

Two-dimensional NMR lineshape analysis of single, multiple, zero and double quantum correlation experiments

NMR spectroscopy provides a powerful approach for the characterisation of chemical exchange and molecular interactions by analysis of series of experiments acquired over the course of a titration measurement. The appearance of NMR resonances undergoing chemical exchange depends on the frequency difference relative to the rate of exchange, and in the case of one-dimensional experiments chemical exchange regimes are well established and well known. However, two-dimensional experiments present additional complexity, as at least one additional frequency difference must be considered. Here we provide a systematic classification of chemical exchange regimes in two-dimensional NMR spectra. We highlight important differences between exchange in HSQC and HMQC experiments, that on a practical level result in more severe exchange broadening in HMQC spectra, but show that complementary alternatives to the HMQC are available in the form of HZQC and HDQC experiments. We present the longitudinal relaxation optimised SOFAST-H(Z/D)QC experiment for the simultaneous acquisition of sensitivity-enhanced HZQC and HDQC spectra, and the longitudinal and transverse relaxation optimised BEST-ZQ-TROSY for analysis of large molecular weight systems. We describe the application of these experiments to the characterisation of the interaction between the Hsp90 N-terminal domain and a small molecule ligand, and show that the independent analysis of HSQC, HMQC, HZQC and HDQC experiments provides improved confidence in the fitted dissociation constant and dissociation rate. Joint analysis of such data may provide improved sensitivity to detect and analyse more complex multi-state interaction mechanisms such as induced fit or conformational selection.

biophysics↗

bioRxiv2019

Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

biophysics↗

bioRxiv2019

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

The over-expression and aggregation of -synuclein (Syn) are linked to the onset and pathology of Parkinsons disease. Native monomeric Syn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of Syn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric Syn is incubated with microchips arrayed with tethered compounds, we identified novel Syn interacting drug-like compounds. Because these small molecules could impact a variety of Syn forms present in the ensemble, we tested representative hits for impact on multiple Syn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits Syn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by Syn overexpression, and a compound blocking cellular transmission of Syn. Our studies demonstrate that drug-like small molecules that interact with native Syn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of Syn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinsons disease.

neuroscience↗

bioRxiv2018

Developing NMR methods for macromolecular machines: Measurement of residual dipolar couplings to probe dynamical regions of the ribosome

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the stalk protein bL12, and this information was used to refine a 3D structure of its C-terminal domain (CTD). Orientational constraints on the CTD alignment arising from the semiflexible linker sequence were further probed by a paramagnetic alignment strategy, and provided direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome-nascent chain complexes.

biophysics↗

bioRxiv2017

Systematic mapping of the free energy landscapes of a growing immunoglobulin domain identifies a kinetic intermediate associated with co-translational proline isomerization

Co-translational folding is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the wasteful or hazardous formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of co-translational folding pathways. Here we contrast observations in translationally-arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome. By using this approach we identify and characterize two folding intermediates, including a partially folded intermediate associated with the isomerization of a conserved proline residue, which, together with measurements of folding kinetics, raises the prospect that neighboring unfolded domains might accumulate during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation, and show that catalysis of folding by peptidyl-prolyl isomerases is essential to eliminate this hazard.

biophysics↗