The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

peer reviewedGenetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis

Suppressed-moment 2- k order in the canonical frustrated antiferromagnet Gd2Ti2O7

In partially ordered magnets, order and disorder coexist in the same magnetic phase, distinct from both spin liquids and spin solids. Here, we determine the nature of partial magnetic ordering in the canonical frustrated antiferromagnet Gd2Ti2O7, in which Gd3+ spins occupy a pyrochlore lattice. Using single-crystal neutron-diffraction measurements in applied magnetic field, magnetic symmetry analysis, inelastic neutron-scattering measurements, and spin-wave modeling, we show that its low-temperature magnetic structure involves two propagation vectors (2-k structure) with suppressed ordered magnetic moments and enhanced spin-wave fluctuations. Our experimental results are consistent with theoretical predictions of thermal fluctuation-driven order in Gd2Ti2O7, and reveal that inelastic neutron-scattering measurements on powder samples can solve the longstanding problem of distinguishing single-k and multi-k magnetic structures

First Crystal Structure of Bacterial Oligopeptidase B in an Intermediate State: The Roles of the Hinge Region Modification and Spermine

Oligopeptidase B (OpB) is a two-domain, trypsin-like serine peptidase belonging to the S9 prolyloligopeptidase (POP) family. Two domains are linked by a hinge region that participates in the transition of the enzyme between two major states—closed and open—in which domains and residues of the catalytic triad are located close to each other and separated, respectively. In this study, we described, for the first time, a structure of OpB from bacteria obtained for an enzyme from Serratia proteomaculans with a modified hinge region (PSPmod). PSPmod was crystallized in a conformation characterized by a disruption of the catalytic triad together with a domain arrangement intermediate between open and closed states found in crystals of ligand-free and inhibitor-bound POP, respectively. Two additional derivatives of PSPmod were crystallized in the same conformation. Neither wild-type PSP nor its corresponding mutated variants were susceptible to crystallization, indicating that the hinge region modification was key in the crystallization process. The second key factor was suggested to be polyamine spermine since all crystals were grown in its presence. The influences of the hinge region modification and spermine on the conformational state of PSP in solution were evaluated by small-angle X-ray scattering. SAXS showed that, in solution, wild-type PSP adopted the open state, spermine caused the conformational transition to the intermediate state, and spermine-free PSPmod contained molecules in the open and intermediate conformations in dynamic equilibrium

Field-induced magnetic states in geometrically frustrated SrEr2O4

We report an unusual in-field behaviour of SrEr2O4 for a magnetic field applied along two high-symmetry directions, the a and c axes. This geometrically frustrated magnet hosts two crystallographically inequivalent Er ions, Er1 and Er2, that are both located on triangular zigzag ladders, but only one site, Er1, forms a long-range magnetic order at low temperatures in a zero field. We follow the sequence of peculiar field induced states in SrEr2O4 with detailed single-crystal magnetisation and neutron diffraction experiments. On application of an external field along the c axis, the long-range antiferromagnetic order of the Er1 ions is rapidly destroyed and replaced, in fields between 2 and 5 kOe, by a state with shorter-range correlations. The change in correlation length coincides with a fast increase in magnetisation during the metamagnetic transition above which a long-range order is reestablished and maintained into the high fields. The high-field ferromagnet-like order is characterised by significantly different magnetic moments on the two Er sites, with the Er1 site dominating the magnetisation process. For the field applied parallel to the a axis, in the field range of 4 to 12 kOe, the planes of diffuse magnetic scattering observed in zero field due to the one-dimensional correlations between the Er2 moments are replaced by much more localised but still diffuse features corresponding to the establishment of an up-up-down structure associated with a one-third magnetisation plateau. Above 14 kOe, a ferromagnet-like high-field order is induced following another phase transition. For this direction of the field, the Er2 moments dictate the succession of transitions while the Er1 moments remain significantly less polarised. A complete field polarisation of both Er sites is not achieved even at 50 kOe for either field direction, reflecting the strongly anisotropic nature of magnetisation process in SrEr2O4

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

Myelin Basic Protein Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data

Elucidation of the Conformational Transition of Oligopeptidase B by an Integrative Approach Based on the Combination of X-ray, SAXS, and Essential Dynamics Sampling Simulation

Oligopeptidase B (OPB) is the least studied group from the prolyl oligopeptidase family. OPBs are found in bacteria and parasitic protozoa and represent pathogenesis factors of the corresponding infections. OPBs consist of two domains connected by a hinge region and have the characteristics of conformational dynamics, which include two types of movements: the bridging/separation of α/β-hydrolase catalytic and β-propeller-regulatory domains and the movement of a loop carrying catalytic histidine, which regulates an assembly/disassembly of the catalytic triad. In this work, an elucidation of the interdomain dynamics of OPB from Serratia proteamaculans (SpOPB) with and without modification of the hinge region was performed using a combination of X-ray diffraction analysis and small-angle X-ray scattering, which was complemented with an essential dynamics sampling (EDS) simulation. The first crystal structure of catalytically deficient SpOPB (SpOPBS532A) with an intact hinge sequence is reported. Similarly to SpOPB with modified hinges, SpOPBS532A was crystallized in the presence of spermine and adopted an intermediate conformation in the crystal lattice. Despite the similarity of the crystal structures, a difference in the catalytic triad residue arrangement was detected, which explained the inhibitory effect of the hinge modification. The SpOPBS532A structure reconstituted to the wild-type form was used as a starting point to the classical MD followed by EDS simulation, which allowed us to simulate the domain separation and the transition of the enzyme from the intermediate to open conformation. The obtained open state model was in good agreement with the experimental SAXS data

Time-Delayed Anticancer Effect of an Extremely Low Frequency Alternating Magnetic Field and Multimodal Protein–Tannin–Mitoxantrone Carriers with Brillouin Microspectroscopy Visualization In Vitro

The effect of an extremely low frequency alternating magnetic field (ELF AMF) at frequencies of 17, 48, and 95 Hz at 100 mT on free and internalized 4T1 breast cancer cell submicron magnetic mineral carriers with an anticancer drug, mitoxantrone, was shown. The alternating magnetic field (100 mT; 17, 48, 95 Hz; time of treatment—10.5 min with a 30 s delay) does not lead to the significant destruction of carrier shells and release of mitoxantrone or bovine serum albumin from them according to the data of spectrophotometry, or the heating of carriers in the process of exposure to magnetic fields. The most optimal set of factors that would lead to the suppression of proliferation and survival of cells with anticancer drug carriers on the third day (in comparison with the control and first day) is exposure to an alternating magnetic field of 100 mT in a pulsed mode with a frequency of 95 Hz. The presence of magnetic nanocarriers in cell lines was carried out by a direct label-free method, space-resolved Brillouin light scattering (BLS) spectrometry, which was realized for the first time. The analysis of the series of integrated BLS spectra showed an increase in the magnetic phase in cells with a growth in the number of particles per cell (from 10 to 100) after their internalization. The safety of magnetic carriers in the release of their constituent ions has been evaluated using atomic absorption spectrometry