LSSmScarlet2 and LSSmScarlet3, Chemically Stable Genetically Encoded Red Fluorescent Proteins with a Large Stokes’ Shift

Red fluorescent proteins with a large Stokes’ shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes’ shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both “water” and “membrane” conformations of the central loop (loop-2) were determined by X-ray crystallography. The “water” conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The “membrane” conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step—the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their “water” NMR models. We found that the both toxins transform their “water” conformation of loop-2 into the “membrane” one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes

Variability in the Spatial Structure of the Central Loop in Cobra Cytotoxins Revealed by X-ray Analysis and Molecular Modeling

Cobra cytotoxins (CTs) belong to the three-fingered protein family and possess membrane activity. Here, we studied cytotoxin 13 from Naja naja cobra venom (CT13Nn). For the first time, a spatial model of CT13Nn with both “water” and “membrane” conformations of the central loop (loop-2) were determined by X-ray crystallography. The “water” conformation of the loop was frequently observed. It was similar to the structure of loop-2 of numerous CTs, determined by either NMR spectroscopy in aqueous solution, or the X-ray method. The “membrane” conformation is rare one and, to date has only been observed by NMR for a single cytotoxin 1 from N. oxiana (CT1No) in detergent micelle. Both CT13Nn and CT1No are S-type CTs. Membrane-binding of these CTs probably involves an additional step—the conformational transformation of the loop-2. To confirm this suggestion, we conducted molecular dynamics simulations of both CT1No and CT13Nn in the Highly Mimetic Membrane Model of palmitoiloleoylphosphatidylglycerol, starting with their “water” NMR models. We found that the both toxins transform their “water” conformation of loop-2 into the “membrane” one during the insertion process. This supports the hypothesis that the S-type CTs, unlike their P-type counterparts, require conformational adaptation of loop-2 during interaction with lipid membranes

Local Flexibility of a New Single-Ring Chaperonin Encoded by Bacteriophage AR9 Bacillus subtilis

Chaperonins, a family of molecular chaperones, assist protein folding in all domains of life. They are classified into two groups: bacterial variants and those present in endosymbiotic organelles of eukaryotes belong to group I, while group II includes chaperonins from the cytosol of archaea and eukaryotes. Recently, chaperonins of a prospective new group were discovered in giant bacteriophages; however, structures have been determined for only two of them. Here, using cryo-EM, we resolved a structure of a new chaperonin encoded by gene 228 of phage AR9 B. subtilis. This structure has similarities and differences with members of both groups, as well as with other known phage chaperonins, which further proves their diversity

Interaction of extended loop 882-892 of Cp with Mpo.

<p>Cp is shown in blue, heavy chain of Mpo in palegreen, light chain of Mpo in pink. Copper ions are shown as orange spheres. Heme is shown in violet, ferrous is shown in gray sphere. (<b>A</b>) Overall view of Cp monomer contact with Mpo monomer. Selected contact is indicated by dash-line oval (<b>B</b>). Zoom of contact area of Cp with the entrance of heme pocket of Mpo. Cp is shown in cartoon; Mpo is shown in cartoon and semi-transparent surface representation. Ends of loop between domain 5 and 6 are marked. <b>(C)</b> Inhibition of ABTS-peroxidase activity of Mpo by synthetic peptide RPYLKVFNPR corresponding to the 883-892 fragment of Cp sequence.</p

Contact areas of Cp with Mpo near 3,4 and 5 domains of Cp.

<p>(<b>A</b>) Contact area near N-terminal of Mpo light-chain and loop 699-720 of Cp. (<b>B</b>) Contact of 699-720 loop of Cp with symmetrical monomer of Mpo. Symmetrical molecule of Cp is not shown for clearance. (<b>C</b>) Contact area near 542-557 and 618-624 loops of Cp. (<b>D</b>) Contact area near <i>p-</i>PD site of Cp (domain 4). Residues M668, W669 and H667 are shown in stick representation.</p

Data collection statistics and refinement for Cp-Mpo complex and Cp with free labile sites.

*<p>Values in parentheses are for highest resolution shell.</p

Cp-Mpo-Lf in solution.

<p>(<b>A</b>) Scattering patterns of ternary complex and individual proteins. (1): Mpo dimer, (2) Cp with glycans, (3) glycosylated Lf, (4) Monomeric binary complex Cp-Lf and (5) ternary complex. Experimental data are denoted by dots, fits computed from the appropriate portions of the SAXS model are shown as red solid lines. The plots are displaced along the logarithmic scale for better visualization. Insert: Guinier plots (ln I <i>versus</i> s²) with the linear fits (straight lines) in the ranges automatically determined by AutoRg (Petoukhov et al., 2012). (<b>B</b>) SAXS models of the ternary complex. Rigid body model is presented by Ca trace whereby Cp and Lf subunits (two copies each) and Mpo dimer are shown in red, blue and green, respectively. The location of sugars is denoted by appropriately colored solid spheres. Bottom view is rotated by 90 degrees around horizontal axis. <i>Ab initio</i> shapes of the ternary complex (top) reconstructed in P2 and of Cp-Lf (bottom) are shown as grey beads. (<b>C</b>) Zoom of Cp-Lf interaction in ribbon and transparent surface representation. One of possible Lf sugar chain conformation (brown sticks and pink semitransparent surface) is shown in stick and surface representation. Lf-N1 is shown in chocolate, Lf-N2 in yellow, Lf-C1 in pink and Lf-C2 in red. Cp is shown in light cyan. Border of domain 1 is highlighted by blue line. Glycosylation site N119 indicates by black arrow.</p

Scheme of influence of Cp, Mpo, Lf on functions of each other due to interactions.

<p>Inhibition is shown by thin black arrows, activation or protection of function is shown by bold grey arrows. *Substrates of Mpo: o-DA, 4-chloro-1-naphol, tetramethylbenzidine, ABTS; **Substrates of Cp: <i>o</i>-DA, <i>p</i>-PD, DOPA.</p