Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving ...site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.
We highlight chemical and biochemical strategies taken to optimise peptide and protein modification using peptide ligases.
The occurrence of ice-nucleating particles (INPs) in our atmosphere has a profound impact on the properties and lifetime of supercooled clouds. To date, the identities, sources and abundances of ...particles capable of nucleating ice at relatively low supercoolings (T > −15 °C) remain enigmatic. While biomolecules such as proteins and carbohydrates have been implicated as important high-temperature INPs, the lack of knowledge on the environmental fates of these species makes it difficult to assess their potential atmospheric impacts. Here we show that such nanoscale ice-nucleating proteins from a common soil-borne fungus (Fusarium avenaceum) preferentially bind to and confer their ice-nucleating properties to kaolinite. The ice-nucleating activity of the proteinaceous INPs is unaffected by adsorption to the clay, and once bound the proteins do not readily desorb, retaining much of the activity even after multiple washings with pure water. The atmospheric implications of the finding that biological residues can confer their ice-nucleating ability to dust particles are discussed.
Sound production capabilities and characteristics in Loricariidae, the largest catfish family, have not been well examined. Sounds produced by three loricariid catfish species, Otocinclus affinis, ...Pterygoplichthys gibbiceps, and Pterygoplichthys pardalis, were recorded. Each of these species produces pulses via pectoral-fin spine stridulation by rubbing the ridged condyle of the dorsal process of the pectoral-fin spine base against a matching groove-like socket in the pectoral girdle. Light and scanning electron microscopy were used to examine the dorsal process of the pectoral-fin spines of these species. Mean distances between dorsal process ridges of O. affinis, P. gibbiceps, and P. pardalis were 53, 161, and 329 μm, respectively. Stridulation sounds occurred during either abduction (type A) or adduction (type B). O. affinis produced sounds through adduction only and P. pardalis through abduction only, whereas P. gibbiceps often produced pulse trains alternating between abduction and adduction. In these species, dominant frequency was an inverse function of sound duration, fish total length, and inter-ridge distance on the dorsal process of the pectoral-fin spine and sound duration increased with fish total length. While stridulation sounds are used in many behavioral contexts in catfishes, the functional significance of sound production in Loricariidae is currently unknown.
SurE is a standalone peptide cyclase essential for the production of surugamide antibiotics. Although SurE catalyses the cyclisation of varied nonribosomal peptides
in vivo
, its substrate ...specificity is poorly understood. To address this issue, an on-resin SurE cyclisation assay was developed and in combination with SNAC thioesters and kinetic measurements was used to define the chemical space of the N-terminal substrate residue.
The N-terminal substrate specificity of the SurE peptide cyclase was elucidated using a combination of on-resin biomemtic substrates and conventional SNAC thioesters.
“Sorting out” N‐terminal labeling: The reversibility of transpeptidase reactions makes protein N‐terminal labeling challenging. Depsipeptide substrates for sortase A release alcohol by‐products, ...which are poor nucleophiles for the reverse reaction, during ligation. Proteins with an unhindered N‐terminal glycine residue can be labeled efficiently with only a minimal excess of the labeling reagent (see scheme).
In bacteria, many genes involved in the biosynthesis of cofactors such as thiamine pyrophosphate (TPP) are regulated by ribo switches, regions in the 5' end of mRNAs to which the cofactor binds, ...thereby affecting translation and/or transcription. TPP riboswitches have now been identified in fungi, in which they alter mRNA splicing. Here, we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an increase in intracellular thiamine and TPP levels. Comparison with Volvox carteri, a related alga, revealed highly conserved regions within introns of these genes. Inspection of the sequences identified TPP riboswitch motifs, and RNA transcribed from the regions binds TPP in vitro. The THI4 riboswitch, but not the promoter region, was found to be necessary and sufficient for thiamine to repress expression of a luciferase-encoding reporter construct in vivo. The pyr1 mutant of C. reinhardtii, which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP. By the use of these ribo switches, thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin.
Abstract
Background
To assess the relationship between self-reported and performance-based visual impairment (VI) and lower extremity physical function.
Methods
Cross-sectional analysis of 2 219 ...Health ABC participants who completed vision testing and the Short Physical Performance Battery (SPPB). Linear regression models used either self-reported (weighted visual function question VFQ score) or performance-based (visual acuity VA, log contrast sensitivity LCS, Frisby stereoacuity SA) to predict SPPB or its components—gait speed, chair stands, or standing balance—with and without covariate adjustment.
Results
Mean age was 73.5 years (range 69–80); 52.4% were female and 37.4% African American. All VI measures were strongly associated with SPPB in unadjusted and adjusted models (p < .001). A self-reported VFQ score 1 standard deviation lower than the mean (mean 87.8 out of 100) demonstrated a −0.241 (95% confidence interval CI: −0.325, −0.156) adjusted difference in SPPB. After controlling for covariates, VA of <20/40 (41%) demonstrated a −0.496 (−0.660, −0.331) lower SPPB score while SA score>85 arcsec (30%) had a −0.449 (−0.627, −0.271) adjusted SPPB score versus those with better visual function. LCS < 1.55 (28.6%) was associated with a −0.759 (−0.938, −0.579) lower and LCS ≤ 1.30 (8%) with a −1.216 (−1.515, −0.918) lower adjusted SPPB score relative to better LCS. In a final multivariable model containing multiple vision measures, LCS remained independently associated with SPPB and all components, while SA remained associated with balance (all p < .05).
Conclusions
Both self-reported and performance-based VI are strongly associated with poor lower extremity physical function. These findings may identify a subgroup of older adults with co-existing visual and physical dysfunction who may benefit from targeted screening and intervention to prevent disability.
X-linked retinitis pigmentosa (XLRP) is genetically heterogeneous with two causative genes identified, RPGR and RP2. We previously mapped a locus for a severe form of XLRP, RP23, to a 10.71 Mb ...interval on Xp22.31-22.13 containing 62 genes. Candidate gene screening failed to identify a causative mutation, so we adopted targeted genomic next-generation sequencing of the disease interval to determine the molecular cause of RP23. No coding variants or variants within or near splice sites were identified. In contrast, a variant deep within intron 9 of OFD1 increased the splice site prediction score 4 bp upstream of the variant. Mutations in OFD1 cause the syndromic ciliopathies orofaciodigital syndrome-1, which is male lethal, Simpson-Golabi-Behmel syndrome type 2 and Joubert syndrome. We tested the effect of the IVS9+706A>G variant on OFD1 splicing in vivo. In RP23 patient-derived RNA, we detected an OFD1 transcript with the insertion of a cryptic exon spliced between exons 9 and 10 causing a frameshift, p.N313fs.X330. Correctly spliced OFD1 was also detected in patient-derived RNA, although at reduced levels (39%), hence the mutation is not male lethal. Our data suggest that photoreceptors are uniquely susceptible to reduced expression of OFD1 and that an alternative disease mechanism can cause XLRP. This disease mechanism of reduced expression for a syndromic ciliopathy gene causing isolated retinal degeneration is reminiscent of CEP290 intronic mutations that cause Leber congenital amaurosis, and we speculate that reduced dosage of correctly spliced ciliopathy genes may be a common disease mechanism in retinal degenerations.
Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer ...assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.
Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies ...remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.
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