Circularly Permuted Far-Red Fluorescent Proteins

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the “optical window” above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

Structure and stability of the designer protein WRAP-T and its permutants

Abstract$$\beta $$ β -Propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats (‘blades’) each consisting of a 4-stranded anti-parallel $$\beta $$ β -sheet. So far, 4- to 12-bladed $$\beta $$ β -propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric $$\beta $$ β -propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each $$\beta $$ β -propeller sub-family.

Design of a Marine Sediments Resistivity Measurement System Based on a Circular Permutation Electrode

Marine sediments are rich in mineral resources, organic resources, and microbial life. The study of marine sediments is of great significance for the development and utilization of marine resources and understanding the life process. Resistivity is the overall characteristic of the electrical conductivity of marine sediments. Measuring the resistivity of marine sediments is helpful to ascertain the marine geological structure, study the distribution of marine mineral resources, and evaluate the marine soil environment. Measuring the resistivity of marine sediments is of great significance to promote marine exploration. At present, the resistivity measurement device on the market can be directly used to measure soil and water on land, but if used to measure marine sediments, it will be disturbed by seawater temperature and pressure, resulting in large errors. In this paper, a high-precision pressure-maintaining transfer system of marine sediment resistivity measurement instrument based on circular permutation electrode is designed, which can measure the resistivity of marine sediment samples after pressure-maintaining transfer. At the same time, a new type of circular permutation electrode measurement method is proposed, which makes the resistivity value more accurate, reduces the length of the probe appropriately, and saves the cost. By measuring the resistivity of marine sediments, the type of sediments can be inverted, which provides a way of thinking about the promotion of the research and development and utilization of marine resources.

Circularly permuted LOV domain as an engineering module for optogenetic tools

We re-engineered a commonly-used light-sensing protein, LOV domain, using a circular permutation strategy to allow photoswitchable control of the C-terminus of a peptide. We demonstrate that the use of circularly permuted LOV domain on its own or together with the original LOV could expand the engineering capabilities of optogenetic tools.

Structure and Stability of the designer protein WRAP-T and its permutants

β-propeller proteins are common natural disc-like pseudo-symmetric proteins that contain multiple repeats ('blades') each consisting of a 4-stranded anti-parallel beta-sheet. So far, 4- to 12-bladed β-propellers have been discovered in nature showing large functional and sequential variation. Using computational design approaches, we created perfectly symmetric β-propellers out of natural pseudo-symmetric templates. These proteins are useful tools to study protein evolution of this very diverse fold. While the 7-bladed architecture is the most common, no symmetric 7-bladed monomer has been created and characterized so far. Here we describe such a engineered protein, based on a highly symmetric natural template, and test the effects of circular permutation on its stability. Geometrical analysis of this protein and other artificial symmetrical proteins reveals no systematic constraint that could be used to help in engineering of this fold, and suggests sequence constraints unique to each β-propeller sub-family.

Plant asparaginyl endopeptidases and their structural determinants of function

Asparaginyl endopeptidases (AEPs) are versatile enzymes that in biological systems are involved in producing three different catalytic outcomes for proteins, namely (i) routine cleavage by bond hydrolysis, (ii) peptide maturation, including macrocyclisation by a cleavage-coupled intramolecular transpeptidation and (iii) circular permutation involving separate cleavage and transpeptidation reactions resulting in a major reshuffling of protein sequence. AEPs differ in their preference for cleavage or transpeptidation reactions, catalytic efficiency, and preference for asparagine or aspartate target residues. We look at structural analyses of various AEPs that have laid the groundwork for identifying important determinants of AEP function in recent years, with much of the research impetus arising from the potential biotechnological and pharmaceutical applications.

Structural basis for a natural circular permutation in proteins

Over 30 years ago, an intriguing post-translational modification was discovered to be responsible for creating concanavalin A (conA), a carbohydrate-binding protein found in the seeds of jack bean (Canavalia ensiformis) and commercially used for carbohydrate chromatography. Biosynthesis of conA involves what was then an unprecedented rearrangement in amino acid sequence, whereby the N-terminal half of the gene-encoded conA precursor is swapped to become the C-terminal half of conA. The cysteine protease, asparaginyl endopeptidase (AEP), was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of conA circular permutation, we generated a recombinant jack bean conA precursor (pro-conA) plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 Å and 2.7 Å respectively. By reconstituting the biosynthesis of conA in vitro, we prove CeAEP1 alone can perform both the cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both these reactions. Biophysical assays illustrated that conA is more thermally and pH stable than pro-conA, consistent with fewer intermolecular interactions between subunits in the pro-conA crystal structure. These findings elucidate the consequences of circular permutation in the only post-translation example known to occur in nature.