ABSTRACT
We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
Subject(s)
Dimerization , Endopeptidases/chemistry , Tetrahydrofolate Dehydrogenase/chemistry , Cell Compartmentation/drug effects , Cell Compartmentation/genetics , Cell Compartmentation/radiation effects , Light , Organelles/chemistry , Organelles/radiation effects , Protein Transport/drug effects , Protein Transport/genetics , Protein Transport/radiation effects , Sirolimus/chemistry , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Tetrahydrofolate Dehydrogenase/radiation effectsABSTRACT
A state-of-the-art review of the role of the Langmuir-Blodgett nanotemplate on protein crystal structures is here presented. Crystals grown by nanostructured template appear more radiation resistant than the classical ones, even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The electron density maps and the changes in parameters such as total diffractive power, B-factor, and pairwise R-factor have been discussed. Protein crystals, grown by the Langmuir-Blodgett nanotemplate-based method, proved to be more radiation resistant compared to crystals grown by the classical hanging drop method in terms of both global and specific damage.
Subject(s)
Crystallization/methods , Nanostructures/chemistry , Nanotechnology/methods , Radiation , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/radiation effects , Dose-Response Relationship, Radiation , Models, Molecular , Proteins/chemistry , Proteins/radiation effects , Synchrotrons/instrumentation , Tetrahydrofolate Dehydrogenase/chemistry , Tetrahydrofolate Dehydrogenase/radiation effectsABSTRACT
An ionizing radiation resistant derivative was obtained from the mouse P19H22 (aprt hemizygote) embryonal carcinoma cell line by repeated exposure to 137Cs gamma radiation. Ionizing radiation resistance in the 6Gy-R cell line was not correlated with a failure to undergo cell cycle arrest or a loss of the p53 response after exposure to 137Cs gamma radiation. Moreover, the cells did not display increased resistance to bleomycin, a double strand break inducing agent. However, the cells did display increased resistance to ultraviolet radiation, ethyl methanesulfonate, and 95% oxygen. A mutational analysis demonstrated a > 700 fold-fold increase in the frequency of aprt mutants for the 6Gy-R cells, but no change in the frequency of hprt or dhfr mutants. A molecular analysis suggested that the aprt mutations in the 6Gy-R cells arose by recombinational events. A possible association between radiation resistance, DNA repair, and a mutator phenotype for large-scale mutational events is discussed.