Biology subjects

Jones, N. C.

Publications and source records attributed to Jones, N. C..

8 recordsLinked to original sources

bioRxiv2023

A Reference Data Set for Circular Dichroism Spectroscopy Comprised of Validated Intrinsically Disordered Protein Models

Circular Dichroism (CD) spectroscopy is an analytical technique that measures the wavelength-dependent differential absorbance of circularly polarized light, and is applicable to most biologically important macromolecules, such as proteins, nucleic acids, and carbohydrates. It serves to characterize the secondary structure composition of proteins, including intrinsically disordered proteins, by analyzing their recorded spectra. Several computational tools have been developed to interpret protein CD spectra. These methods have been calibrated and tested mostly on globular proteins with well-defined structures, mainly due to the lack of reliable reference structures for disordered proteins. It is therefore still largely unclear how accurately these computational methods can determine the secondary structure composition of disordered proteins. Here, we provide such a required reference data set consisting of model structural ensembles and matching CD spectra for eight intrinsically disordered proteins. Using this set of data, we have assessed the accuracy of several published CD prediction and secondary structure estimation tools, including our own CD analysis package SESCA. Our results show that for most of the tested methods, their accuracy for disordered proteins is generally lower than for globular proteins. In contrast, SESCA, which was developed using globular reference proteins, but was designed to be applicable to disordered proteins as well, performs similarly well for both classes of proteins. The new reference data set for disordered proteins should allow for further improvement of all published methods.

biophysics↗

bioRxiv2023

Conformational analysis of membrane-proximal segments of GDAP1 in a lipidic environment using synchrotron radiation suggests a mode of assembly at the mitochondrial outer membrane

The mitochondrial outer membrane creates a diffusion barrier between the cytosol and mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflet of the mitochondrial outer membrane in different orientations. Impairment of such interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain.

biochemistry↗

bioRxiv2022

Mouse behaviour on the trial-unique non-matching-to-location (TUNL) touchscreen task reflects a mixture of distinct working memory codes and response biases

The trial-unique non-matching to location (TUNL) touchscreen task shows promise as a translational assay of working memory deficits in disorders including autism, ADHD, and schizophrenia. Although it is commonly assumed that the TUNL task predominantly measures spatial working memory in rodents, this proposition has not previously been tested. In this project, we used computational modelling of behaviour from mice performing the TUNL task (total N = 163 mice across three datasets; 158,843 total trials). Contrary to common assumptions, behaviour on the TUNL task did not exclusively reflect spatial working memory. Instead, choice behaviour was explained as a mixture of both retrospective (spatial) working memory and prospective working memory for an intended behavioural response, as well as animal-specific response biases. We suggest that these findings can be understood within a resource-rational cognitive framework, and propose several task-design principles that we predict will maximise spatial working memory and minimise alternative behavioural strategies.

animal behavior and cognition↗

bioRxiv2022

Structure and Protein-Protein Interactions of Ice Nucleation Proteins Drive Their Activity

ABSTRACTMicrobially-produced ice nucleating proteins (INpro) are unique molecular structures with the highest known catalytic efficiency for ice formation. Their critical role in rain formation and frost damage of crops together with their diverse commercial applications warrant an in-depth under-standing of their inherent ice nucleation mechanism. We used the machine-learning based software Al-phaFold to develop the first ab initio structural model of a bacterial INpro which is a novel beta-helix structure consisting of repeated stacks of two beta strands connected by two sharp turns. Using the synchrotron radiation circular dichroism, we validated the {beta}-strand content of the model. Combining functional studies of purified recombinant INpro, electron microscopy and modeling, we further demonstrate that the formation of dimers and higher-order oligomers is key to INpro activity. This work presents a major advance in understanding the molecular foundation for bacterial ice-nucleation activity and the basis for investigating the mechanistic role of INpro-induced ice formation in the atmosphere, and for commercial design and production of ice-nucleating particles for industrial applications.

microbiology↗

bioRxiv2019

Helix formation during the coupled binding and folding of intrinsically disordered proteins monitored by synchrotron-radiation circular dichroism spectroscopy

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signalling processes. These proteins often gain structure upon binding to their target proteins in multi-step reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain (NCBD) and the disordered interaction domain from activator of thyroid hormone and retinoid receptors (ACTR). We demonstrate that helix formation in a rapid binding reaction can be followed by stopped flow synchrotron-radiation circular dichroism spectroscopy, and describe the design of such a beamline. Fluorescence-monitored binding experiments of ACTR and NCBD display several kinetic phases including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time resolved circular dichroism experiments show that almost all helicity is formed upon initial association of the proteins, or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron radiation circular dichroism for mechanistic studies of protein-protein interactions.

biophysics↗

bioRxiv2019

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment.

biophysics↗

bioRxiv2019

Ionic strength and calcium regulate the membrane interactions of myelin basic protein and the cytoplasmic domain of myelin protein zero

The formation of a mature myelin sheath in the vertebrate nervous system requires specific protein-membrane interactions. Several myelin-specific proteins are involved in the stacking of lipid membranes into multilayered structures around neuronal axons, and misregulation of these processes may contribute to chronic demyelinating diseases. Two key proteins functioning in myelin membrane binding and stacking are the myelin basic protein (MBP) and protein zero (P0). Other factors, including Ca2+, are important for the regulation of myelination. Here, we studied the effects of ionic strength and Ca2+ on the direct molecular membrane interactions of MBP and the cytoplasmic domain of P0 (P0ct). While both MBP and P0ct bound and aggregated negatively charged lipid vesicles, while simultaneously folding, both ionic strength and calcium had systematic effects on these interactions. Especially when decreasing membrane net negative charge, the level and kinetics of vesicle aggregation, which is a functional assay for myelin membrane-stacking proteins, were affected by both salt and Ca2+. The results indicate that the effects on lipid membrane surfaces by ions can directly affect myelin protein-membrane interactions at the molecular level, in addition to signalling effects in myelinating glia.

biochemistry↗

bioRxiv2018

SESCA: Predicting the Circular Dichroism Spectra of Proteins from Molecular Structure

Circular dichroism spectroscopy is a highly sensitive, but low-resolution technique to study the structure of proteins. Combined with molecular modelling or other complementary techniques, CD spectroscopy can provide essential information at higher resolution. To this end, we introduce a new computational method to calculate the electronic circular dichroism spectra of proteins from a structural model or ensemble using the average secondary structure composition and a pre-calculated set of basis spectra. We compared the predictive power of our method to existing algorithms - namely DichroCalc and PDB2CD - and found that it predicts CD spectra more accurately, with a 50% smaller average deviation from the measured CD spectra. Our results indicate that the derived basis sets are robust to experimental errors in the reference spectra and to the choice of the secondary structure classification algorithm. For over 80% of the globular reference proteins, our basis sets accurately predict the experimental spectrum solely from their secondary structure composition. For the remaining 20%, correcting for intensity normalization considerably improves the prediction power. Additionally, we show that the predictions for short peptides and intrinsically disordered proteins strongly benefit from accounting for side-chain contributions and structural flexibility. Table Of Content Graphics O_TBL View this table: org.highwire.dtl.DTLVardef@1a1d73forg.highwire.dtl.DTLVardef@3aaa8forg.highwire.dtl.DTLVardef@193c090org.highwire.dtl.DTLVardef@1474f92org.highwire.dtl.DTLVardef@b920da_HPS_FORMAT_FIGEXP M_TBL C_TBL

biophysics↗