Comparative structural analysis of a histone-like protein from Spiroplasma melliferum in the crystalline state and in solution

A solution of a histone-like protein from Spiroplasma melliferum (HUSpm) was examined by small-angle X-ray scattering (SAXS). The experimental SAXS curve was compared with those calculated for the HUSpm structures from the PDB databank obtained by both X-ray diffraction analysis and nuclear magnetic resonance spectroscopy. The model of the HUSpm structure in solution, which best agrees with the experimental SAXS data, has a shorter distance between the centers of mass of the HUSpm monomers compared to the crystal structure, indicating that the HUSpm monomers can be located closer to each other in solution than in the crystalline state

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

The Crystal Structure of Nα-p-tosyl-lysyl Chloromethylketone-Bound Oligopeptidase B from Serratia Proteamaculans Revealed a New Type of Inhibitor Binding

A covalent serine protease inhibitor—Na-p-Tosyl-Lysyl Chloromethylketone (TCK) is a modified lysine residue tosylated at the N-terminus and chloromethylated at the C-terminus, one molecule of which is capable of forming two covalent bonds with both Ser and His catalytic residues, was co-crystallized with modified oligopeptidase B (OpB) from Serratia proteomaculans (PSPmod). The kinetics study, which preceded crystallization, shows that the stoichiometry of TCK-dependent inhibition of PSPmod was 1:2 (protein:inhibitor). The crystal structure of the PSPmod-TCK complex, solved at a resolution of 2.3 Å, confirmed a new type of inhibitor binding. Two TCK molecules were bound to one enzyme molecule: one with the catalytic Ser, the other with the catalytic His. Due to this mode of binding, the intermediate state of PSPmod and the disturbed conformation of the catalytic triad were preserved in the PSPmod-TCK complex. Nevertheless, the analysis of the amino acid surroundings of the inhibitor molecule bound to the catalytic Ser and its comparison with that of antipain-bound OpB from Trypanosoma brucei provided an insight in the structure of the PSPmod substrate-binding pocket. Supposedly, the new type of binding is typical for the interaction of chloromethylketone derivatives with two-domain OpBs. In the open conformational state that these enzymes are assumed in solution, the disordered configuration of the catalytic triad prevents simultaneous interaction of one inhibitor molecule with two catalytic residues

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis

YTnC2, an improved genetically encoded green calcium indicator based on toadfish troponin C

Genetically encoded calcium indicators based on truncated troponin C are attractive probes for calcium imaging due to their relatively small molecular size and twofold reduced calcium ion buffering. However, the best‐suited members of this family, YTnC and cNTnC, suffer from low molecular brightness, limited dynamic range, and/or poor sensitivity to calcium transients in neurons. To overcome these limitations, we developed an enhanced version of YTnC, named YTnC2. Compared with YTnC, YTnC2 had 5.7‐fold higher molecular brightness and 6.4‐fold increased dynamic range in vitro. YTnC2 was successfully used to reveal calcium transients in the cytosol and in the lumen of mitochondria of both mammalian cells and cultured neurons. Finally, we obtained and analyzed the crystal structure of the fluorescent domain of the YTnC2 mutant

Comparison of histone-like HU protein DNA-binding properties and HU/IHF protein sequence alignment

<div><p>Background</p><p>The structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA–binding properties and other protein features.</p><p>Methodology and principal findings</p><p>Performed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA–binding properties of HU from <i>N</i>. <i>gonorrhoeae</i>, which sequence is similar to one of <i>E</i>.<i>coli</i> HU, and HU from <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> which sequences are distant from <i>E</i>.<i>coli</i> protein. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU essentially differs from <i>E</i>.<i>coli</i> HU. In respect to binding of distorted DNA structures, <i>S</i>. <i>melliferum</i> HU and <i>E</i>.<i>coli</i> HU have similar properties but essentially different from <i>M</i>. <i>gallisepticum</i> HU and <i>N</i>. <i>gonorrhea</i> HU. We found that in respect to dsDNA binding, only <i>S</i>. <i>melliferum</i> HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than <i>E</i>.<i>coli</i> and <i>N</i>. <i>gonorrhoeae</i>. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.</p><p>Conclusions and significance</p><p>HU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding’s even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from <i>N</i>. <i>gonorrhoeae</i>, <i>M</i>. <i>gallisepticum</i> and <i>S</i>. <i>melliferum</i> are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.</p></div

Model of HU <i>Bifidobacterium longum</i> from Actinobacteria (clade HU_acti_0), superimposed with <i>E</i>. <i>coli</i> HUα model.

<p><i>B</i>. <i>longum</i> HU is shown in yellow, <i>E</i>. <i>coli</i> HUα—in cyan. Angle between alpha helix 2 and alpha helix 3 is shown in magenta.</p

Consensus sequences of three IHF_A, three IHF_B, and several HU clades revealed in this study.

<p>Conservative residues are highlighted. Alpha helixes and beta sheets are indicated with yellow and blue blocks, respectively.</p

PCA plot of the location of aligned sequences of HU/IHF proteins on three axes.

<p>Three main groups of proteins, HU, IHF_A and IHF_B, are indicated as well as results of further subdivision of the protein sequences (HU and IHF clades).<b>A.</b> Most populated HU clades: clade HU_Firmicutes (mainly originated from HU of Firmicutes species) and clades HU_ecoA and HU_ecoB (mainly from proteobacteria) are shown in magenta, bleu and cyan, respectively. Other apparent HU/IHF clades are indicated. Position HUs and IHFs proteins of <i>E</i>. <i>coli</i> are shown in red, as well as positions of HU of <i>N</i>. <i>gonorrhoeae</i> (NG), <i>M</i>. <i>gallisepticum</i> (MG), and <i>S</i>. <i>melliferum</i> (SM). <i>E</i>. <i>coli</i> HUα and Huβ are very close to each other (62 identities in 90 amino acid core sequence), and IHFα and IHFβ are far from each other (24 identities of 90). We believe that it is the reason why HU separation onto two groups, one close to HUα, and another close to HUβ, is ambiguous. <b>B.</b> IHF clades. Result of two major IHF group subdivisions shown in color: IHF_A magenta, cyan and green, IHF_B red, yellow, and blue. HU sequences are shown in grey.</p