Biology subjects

Menzies, G.

Publications and source records attributed to Menzies, G..

5 recordsLinked to original sources

bioRxiv2025

Structure, function and dynamics of mCoral, a pH responsive engineered variant of the mCherry fluorescent protein with improved hydrogen peroxide tolerance.

The red fluorescent protein mCherry is one of the most utilised fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant termed mCoral due to its orange fluorescence hue has similar brightness to mCherry but has improved resistance to hydrogen peroxide. The variant is also responsive to pH with a low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the {beta}-barrel structure that is filled by a water molecule, which makes new polar interactions including with backbone carbonyl group of F65. Molecular dynamic simulations suggests that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore; the mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral potentially explaining the increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining structure-function relationship in red fluorescent proteins.

biochemistry↗

bioRxiv2023

Developing small Cas9 hybrids using molecular modeling

The RNA-guided CRISPR-Cas9 from Streptococcus pyogenes is the best characterized enzyme for gene editing. Its large size, however, precludes it from being packaged together with its single guide (sg)RNA into a single adeno-associated virus, limiting in vivo applications. Here, we developed smaller Cas9 hybrids, made of the PAM interacting domain (PID) of S. pyogenes and the catalytic domains of the smaller Cas9 orthologues, as well as sgRNA cognate hybrids. Molecular modeling revealed that the presence of a sgRNA stabilizes Cas9. Making the D10A mutation to turn Cas9 into a nickase dramatically alters its binding energy to the sgRNA, showing that the approach can identify functionally relevant changes. However, we found that the four Cas9/sgRNA hybrid pairs tested in human cells failed to edit target sequences. We conclude that in silico approaches can identify functional changes caused by point mutations but are not sufficient for designing Cas9/sgRNA hybrids.

bioengineering↗

bioRxiv2022

N-linked glycosylation increases horse radish peroxidase rigidity leading to enhanced activity and stability.

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here we show glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its activity and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo-HRPs thermal stability and promoted significant helical structure in the absence of haem (apo-HRP). Glycosylation also resulted in a 10-fold increase in enzymatic turnover towards o-phenylenediamine dihydrochloride when compared to its non-glycosylated form. Utilising a naturally occurring site-specific probe of active site flexibility (Trp117) in combination with red-edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data shows that glycosylation does not just have a passive effect on HRP stability but can exert long range effects that mediate the native enzymes activity and stability through changes in inherent dynamics.

biochemistry↗

bioRxiv2021

FAN1 nuclease activity affects CAG expansion and age at onset of Huntington's disease

The age at onset of motor symptoms in Huntingtons disease (HD) is driven by HTT CAG repeat length but modified by other genes. We used exome sequencing of 683 HD patients with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We identified damaging coding variants in candidate modifier genes from prior genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier onset HD. Nuclease activities of these variants correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion comparable to those observed with complete FAN1 knock out. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

neuroscience↗

bioRxiv2019

Genetic association study of psychotic experiences in UK Biobank

Psychotic experiences, such as hallucinations and delusions, are reported by approximately 5%-10% of the general population, though only a small proportion of individuals develop psychotic disorders such as schizophrenia or bipolar disorder. Studying the genetic aetiology of psychotic experiences in the general population, and its relationship with the genetic aetiology of other disorders, may increase our understanding of their pathological significance. Using the population-based UK Biobank sample, we performed the largest genetic association study of psychotic experiences in individuals without a psychotic disorder. We conducted three genome-wide association studies (GWAS) for (i) any psychotic experience (6123 cases vs. 121,843 controls), (ii) distressing psychotic experiences (2143 cases vs. 121,843 controls), and (iii) multiple occurrence psychotic experiences (3337 cases vs. 121,843 controls). Analyses of polygenic risk scores (PRS), genetic correlation, and copy number variation (CNV) were conducted to assess whether genetic liability to psychotic experiences is shared with schizophrenia and/or other neuropsychiatric disorders and traits. GWAS analyses identified four loci associated with psychotic experiences including a locus in Ankyrin-3 (ANK3, OR=1.16, p=3.06 x 10-8) with any psychotic experience and a locus in cannabinoid receptor 2 gene (CNR2, OR=0.66, p=3.78x10-8) with distressing psychotic experiences. PRS analyses identified associations between psychotic experiences and genetic liability for schizophrenia, major depressive disorder, and bipolar disorder, and these associations were stronger for distressing psychotic experiences. Genetic correlation analysis identified significant genetic correlations between psychotic experiences and major depressive disorder, schizophrenia, autism spectrum disorder and a cross-disorder GWAS. Individuals reporting psychotic experiences had an increased burden of CNVs previously associated with schizophrenia (OR=2.04, p=2.49x10-4) and of those associated with neurodevelopmental disorders more widely (OR=1.75, p=1.41x10-3). In conclusion, we identified four genome-wide significant loci in the largest GWAS of psychotic experiences from the population-based UK Biobank sample and found support for a shared genetic aetiology between psychotic experiences and schizophrenia, but also major depressive disorder, bipolar disorder and neurodevelopmental disorders.

genetics↗