The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757408 | PMC |
| http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148256 | PLOS |
Publication Analysis
Top Keywords
Similar Publications
Magnetosensitivity of tightly bound radical pairs in cryptochrome is enabled by the quantum Zeno effect.
Nat Commun
December 2024
Living Systems Institute, University of Exeter, Stocker Road, Exeter, Devon, EX4 4QD, UK.
The radical pair mechanism accounts for the magnetic field sensitivity of a large class of chemical reactions and is hypothesised to underpin numerous magnetosensitive traits in biology, including the avian compass. Traditionally, magnetic field sensitivity in this mechanism is attributed to radical pairs with weakly interacting, well-separated electrons; closely bound pairs were considered unresponsive to weak fields due to arrested spin dynamics. In this study, we challenge this view by examining the FAD-superoxide radical pair within cryptochrome, a protein hypothesised to function as a biological magnetosensor.
View Article and Find Full Text PDFComparison of ELISA and IFAT for Leishmania infantum by European and Middle Eastern diagnostic laboratories.
Parasit Vectors
December 2024
College of Public Health, University of Iowa, Iowa City, IA, USA.
Background: Visceral leishmaniosis (VL) is the most severe form of human leishmaniosis, with an estimated 95% case fatality if left untreated. Dogs act as peridomestic reservoir hosts for the protozoan parasite Leishmania infantum, a causative agent for human leishmaniosis, endemic throughout the Mediterranean basin. To assure consistent and accurate surveillance of canine infection and prevent transmission to people, consistent diagnosis of canine L.
View Article and Find Full Text PDFEffective management of pre-existing biofilms using UV-LED through inactivation, disintegration and peeling.
J Hazard Mater
December 2024
Department of Electronic Engineering, Laboratory of Micro/Nano-Optoelectronics, Xiamen University, Xiamen, Fujian 361005, China; Institute of Nanoscience and Applications (INA), Southern University of Science and Technology (SUSTech), Shenzhen 518055, China.
Managing undesirable biofilms is a persistent challenge in water treatment and distribution systems. Although ultraviolet-light emitting diode (UV-LED) irradiation, an emerging disinfection method with the chemical-free and emission-adjustable merits, has been widely reported effective to inactivate planktonic bacteria, few studies have examined its effects on biofilms. This study aims to fill this gap by exploring the performance and mechanism of UV-LEDs on the prefabricated Escherichia coli (E.
View Article and Find Full Text PDFEngineering of the genetic code.
Curr Opin Biotechnol
December 2024
Department of Life Sciences, Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 8410501, Israel. Electronic address:
The genetic code is a universally conserved mechanism that translates genetic information into proteins, consisting of 64 codons formed by four nucleotide bases. With a few exceptions, the genetic code universally encodes 20 canonical amino acids (AAs) and three stop signals, with many AAs represented by multiple codons. Genetic engineering has expanded this system through approaches like codon reassignment and synthetic base pair introduction, allowing for the incorporation of noncanonical AAs (ncAAs) into proteins, known as genetic code expansion (GCE).
View Article and Find Full Text PDFComplex Genetic Evolution and Treatment Challenges in Myeloid Neoplasms: A Case of Persistent t(2;3)(p15~23;q26)/ Rearrangement, Mutation, and Transient Chimera.
Cancer Genomics Proteomics
December 2024
Section for Cancer Cytogenetics, Institute for Cancer Genetics and Informatics, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway;
Background/aim: Myelodysplastic syndromes (MDSs) are clonal bone marrow disorders characterized by ineffective hematopoiesis. They are classified based on morphology and genetic alterations, with SF3B1 variants linked to favorable prognosis and MECOM rearrangements associated with poor outcomes. The combined effects of these alterations remain unclear.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!