The
development of an efficient water oxidation catalyst is crucial in
the framework of constructing an artificial photo(electro)synthetic
apparatus for the production of solar fuels. Herein, new hydroxy–pyridine–carboxylate
iridium complexes are reported exhibiting high activity in water oxidation
with both cerium ammonium nitrate and NaIO4 as sacrificial
oxidants. With the latter, the catalytic activity strongly depends
on the pH and position of the OH-substituent in the pyridine ring,
reaching a record turnover frequency of 458 min–1 and turnover number (>14 500) limited only by the amount
of NaIO4. Kinetic experiments measuring O2 evolution
paralleled by NMR studies on oxidative transformation with NaIO4 suggest that Cp* of the catalyst is readily degraded, whereas
the hydroxy–pyridine–carboxylate ligands remain coordinated
at iridium, tuning its activity.
This
work introduces a technology that combines fluorescence anisotropy
decay with microscale-volume viscometry to investigate the compaction
and dynamics of ribosome-bound nascent proteins. Protein folding in
the cell, especially when nascent chains emerge from the ribosomal
tunnel, is poorly understood. Previous investigations based on fluorescence
anisotropy decay determined that a portion of the ribosome-bound nascent
protein apomyoglobin (apoMb) forms a compact structure. This work,
however, could not assess the size of the compact region. The combination
of fluorescence anisotropy with microscale-volume viscometry, presented
here, enables identifying the size of compact nascent-chain subdomains
using a single fluorophore label. Our results demonstrate that the
compact region of nascent apoMb contains 57–83 amino acids
and lacks residues corresponding to the two native C-terminal helices.
These amino acids are necessary for fully burying the nonpolar residues
in the native structure, yet they are not available for folding before
ribosome release. Therefore, apoMb requires a significant degree of
post-translational folding for the generation of its native structure.
In summary, the combination of fluorescence anisotropy decay and microscale-volume
viscometry is a powerful approach to determine the size of independently
tumbling compact regions of biomolecules. This technology is of general
applicability to compact macromolecules linked to larger frameworks.
Proteins are particularly prone to aggregation immediately after release from the ribosome, and it is therefore important to elucidate the role of chaperones during these key steps of protein life. The Hsp70 and trigger factor (TF) chaperone systems interact with nascent proteins during biogenesis and immediately post-translationally. It is unclear, however, whether these chaperones can prevent formation of soluble and insoluble aggregates. Here, we address this question by monitoring the solubility and structural accuracy of globin proteins biosynthesized in an Escherichia coli cell-free system containing different concentrations of the bacterial Hsp70 and TF chaperones. We find that Hsp70 concentrations required to grant solubility to newly synthesized proteins are extremely sensitive to client-protein sequence. Importantly, Hsp70 concentrations yielding soluble client proteins are insufficient to prevent formation of soluble aggregates. In fact, for some aggregation-prone protein variants, avoidance of soluble-aggregate formation demands Hsp70 concentrations that exceed cellular levels in E. coli. In all, our data highlight the prominent role of soluble aggregates upon nascentprotein release from the ribosome and show the limitations of the Hsp70 chaperone system in the case of highly aggregation-prone proteins. These results demonstrate the need to devise better strategies to prevent soluble-aggregate formation upon release from the ribosome.
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