Entity

Time filter

Source Type

Belfast, United Kingdom

Atcheson E.,Queens University of Belfast | Hamilton E.,Queens University of Belfast | Hamilton E.,Belfast Metropolitan College | Pathmanathan S.,Queens University of Belfast | And 4 more authors.
Bioscience Reports | Year: 2011

The IQGAP [IQ-motif-containing GAP (GTPase-activating protein)] family members are eukaryotic proteins that act at the interface between cellular signalling and the cytoskeleton. As such they collect numerous inputs from a variety of signalling pathways. A key binding partner is the calcium-sensing protein CaM (calmodulin). This protein binds mainly through a series of IQ-motifs which are located towards the middle of the primary sequence of the IQGAPs. In some IQGAPs, these motifs also provide binding sites for CaM-like proteins such as myosin essential light chain and S100B. Using synthetic peptides and native gel electrophoresis, the binding properties of the IQ-motifs from human IQGAP2 and IQGAP3 have been mapped. The second and third IQ-motifs in IQGAP2 and all four of the IQ-motifs of IQGAP3 interacted with CaM in the presence of calcium ions. However, there were differences in the type of interaction: while some IQ-motifs were able to form complexes with CaM which were stable under the conditions of the experiment, others formed more transient interactions. The first IQ-motifs from IQGAP2 and IQGAP3 formed transient interactions with CaM in the absence of calcium and the first motif from IQGAP3 formed a transient interaction with the myosin essential light chain Mlc1sa. None of these IQ-motifs interacted with S100B. Molecular modelling suggested that all of the IQ-motifs, except the first one from IQGAP2 formed α-helices in solution. These results extend our knowledge of the selectivity of IQ-motifs for CaM and related proteins. ©The Authors Journal compilation ©2011 Biochemical Society.


Andrews W.J.,Queens University of Belfast | Bradley C.A.,Queens University of Belfast | Hamilton E.,Queens University of Belfast | Hamilton E.,Belfast Metropolitan College | And 3 more authors.
Molecular and Cellular Biochemistry | Year: 2012

IQGAPs are cytoskeletal scaffolding proteins which collect information from a variety of signalling pathways and pass it on to the microfilaments and microtubules. There is a well-characterised interaction between IQGAP and calmodulin through a series of IQ-motifs towards the middle of the primary sequence. However, it has been shown previously that the calponin homology domain (CHD), located at the N-terminus of the protein, can also interact weakly with calmodulin. Using a recombinant fragment of human IQGAP1 which encompasses the CHD, we have demonstrated that the CHD undergoes a calcium ion-dependent interaction with calmodulin. The CHD can also displace the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulphonate from calcium-calmodulin, suggesting that the interaction involves non-polar residues on the surface of calmodulin. Molecular modelling identified a possible site on the CHD for calmodulin interaction. The physiological significance of this interaction remains to be discovered. © 2012 Springer Science+Business Media, LLC.


Magill D.J.,Queens University of Belfast | Hamilton E.,Queens University of Belfast | Hamilton E.,Belfast Metropolitan College | Shirran S.L.,University of St. Andrews | And 3 more authors.
Protein and Peptide Letters | Year: 2016

IQGAPs are eukaryotic proteins which integrate signals from various sources and pass these on the cytoskeleton. Understanding how they do this requires information on the interfaces between the proteins. Here, it is shown that the calponin homology domain of human IQGAP1 (CHD1) can be crosslinked with a-actin. The stoichiometry of the interaction was 1:1. A molecular model was built of the complex and associated bioinformatics analyses predicted that the interaction is likely to involve an electrostatic interaction between Lys-240 of a-actin and Glu-30 of CHD1. These residues are predicted to be accessible and are not involved in many intra-protein interactions; they are thus available for interaction with binding partners. They are both located in regions of the proteins which are predicted to be flexible and disordered; interactions between signalling molecules often involve flexible, disordered regions. The predicted binding region in CHD1 is well conserved in many eukaryotic IQGAP-like proteins. In some cases (e.g Dictyostelium discoideum and Saccharomyces cerevisiae) protein sequence conservation is weak, but molecular modelling reveals that a region of charged, polar residues in a flexible N-terminus is structurally well conserved. Therefore we conclude that the calponin homology domains of IQGAP1-like proteins interact initially through the electrostatic interaction identified here and that there may be subsequent conformational changes to form the final complex. © 2016 Bentham Science Publishers.


Grant
Agency: GTR | Branch: Innovate UK | Program: | Phase: Knowledge Transfer Partnership | Award Amount: 145.20K | Year: 2015

To design and manufacture a new product range from advanced materials to improve competitiveness in global markets for bulk materials conveyance systems and to improve processes to improve cost and time efficiency and reduce waste.


McPherson P.A.C.,Belfast Metropolitan College | Turemen B.T.,Belfast Metropolitan College
Biochemical and Biophysical Research Communications | Year: 2014

Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydroxyl-l-phenylalanine (l-DOPA), which is formed on 3′-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-bound l-DOPA employ lengthy acid hydrolysis steps (ca. 16 h) which may cause artifactual protein oxidation, followed by HPLC with detection based on the intrinsic fluorescence of l-DOPA. We report a novel method for the measurement of protein-bound l-DOPA which involves rapid hydrolysis followed by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The derivatized material is resolved by reversed-phase HPLC in less than 30 min and has derivatization chemistry compatible with both UV and fluorescent detection, providing detection down to the femtomole level. The method provides identical results to those found with highly specific ELISA-based techniques and requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the fast and sensitive nature of the assay will be appealing to those who require large sample throughput. © 2014 Elsevier Inc. All rights reserved.

Discover hidden collaborations