. (2014) Protein secondary structure prediction from circular dichroism spectra using a self-organizing map with concentration correction. Chirality, 26 (9). pp. 471-482. Permanent WRAP url:http://wrap.warwick.ac.uk/75651 Copyright and reuse:The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.Copies of full items can be used for personal research or study, educational, or not-for profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Publisher's statement:"This is the peer reviewed version of the following article: Hall V., Sklepari M. and Rodger A. (2014), Protein Secondary Structure Prediction from Circular Dichroism Spectra Using a Self-Organizing Map with Concentration Correction, Chirality, 471-482, DOI: 10.1002/chir.22338, which has been published in final form at http://dx.doi.org/10.1002/chir.22338. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." A note on versions:The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher's version. Please see the 'permanent WRAP url' above for details on accessing the published version and note that access may require a subscription. ABSTRACT Collecting circular dichroism (CD) spectra for protein solutions is a simple experiment, yet reliable extraction of secondary structure content is dependent on knowledge of the concentration of the protein-which is not always available with accuracy. We previously developed a self-organising map (SOM), called Secondary Structure Neural Network (SSNN), to cluster a database of CD spectra and use that map to assign the secondary structure content of new proteins from CD spectra. The performance of SSNN is at least as good as other available protein CD structure fitting algorithms. In this work we apply SSNN to a collection of spectra of experimental samples where there was suspicion that the nominal protein concentration was incorrect. We show that by plotting the normalized root mean square deviation of the SSNN predicted spectrum from the experimental one versus a concentration scaling-factor it is possible to improve the estimate of the protein concentration while providing an estimate of the secondary structure. For our implementation (51 data points 240 -190 nm in nm increments) good fits and structure estimates are obtained if the NRMSD (normalised root mean square displacement, RMSE/data range) is < 0....
Combination of four well-established techniques complemented with temperature dependence for probing structural changes and detecting differences between insulin samples.
Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications.
A series of new pyrazolo[3,4-c]pyridines bearing various 1, 3, 5 or 1, 3, 7 pattern substitutions, were designed and synthesized. Some of them showed interesting inhibitory activity mainly against glycogen synthase kinase 3 (GSK3)α/β as well as against cdc2-like kinases 1 (CLK1) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), with good selectivity and remarkable structure-activity relationships (SARs), without being cytotoxic. Molecular simulations in correlation with biological data revealed the importance of the existence of N1-H as well as the absence of a bulky 7-substituent.Key words pyrazolopyridine; kinase inhibition; glycogen synthase kinase 3 (GSK3)α/β; purine analogue; molecular simulation Neurodegenerative diseases are among the most challenging diseases with poorly known mechanism and lack of complete cure. Alzheimer's disease (AD) in particular, is the most prevalent cause of dementia, very devastating for the patients and their families, provided that current treatments offer only modest symptomatic relief. This severe mental disorder is mostly aging-associated and represents a global health problem, since it currently affects more than 30 million people worldwide, and its incidence is predicted to rise significantly, due to the increasing average life span. 1) In an effort to explain the pathogenesis of AD, many hypotheses have been explored, among them neurotransmitter modulation, chronic inflammation, metal-induced oxidative stress, elevated cholesterol, and glycogen synthase kinase 3 (GSK-3) implication are of major importance.2) GSK-3 is a ubiquitous serine/threonine kinase, which was first described as the major regulator of glycogen metabolism. It was revealed that GSK-3 plays central roles in important cell signaling pathways, and its malfunction is associated with neurodegeneration and the pathogenesis of several diseases, including diabetes type II, immune and bipolar disorders, chronic inflammation, heart failure and cancer.3) In AD, GSK-3 promotes neuronal death and is a linker between the two histopathological hallmarks: the deposition of extracellular senile plaques composed of amyloid-beta (Aβ) peptide, and the accumulation of hyperphosphorylated microtubule-binding tau protein, leading to tau aggregation and the formation of intracellular neurofibrillary tangles. 4)On the other hand, substantial evidence exists to support the involvement of additional phylogenetically and physiologically relevant protein kinases in neurodegenerative disorders, and, most notably, cyclin dependent kinase 5 (CDK5), 5) dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK 1A), 6) casein kinase 1 (CK1) 7) and cdc2-like kinases CLK1 and CLK2 8) are also investigated as potential targets for the development of novel AD therapeutics. Consequently, the exploitation of small molecule inhibitors for potential inhibitory activity against a set of the above mentioned protein kinases could offer interesting information and assist in the understanding of AD and related tau...
Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that ∼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology.
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