De novo enzyme design has sought to introduce active sites and substrate-binding pockets that are predicted to catalyse a reaction of interest into geometrically compatible native scaffolds1,2, but has been limited by a lack of suitable protein structures and the complexity of native protein sequence–structure relationships. Here we describe a deep-learning-based ‘family-wide hallucination’ approach that generates large numbers of idealized protein structures containing diverse pocket shapes and designed sequences that encode them. We use these scaffolds to design artificial luciferases that selectively catalyse the oxidative chemiluminescence of the synthetic luciferin substrates diphenylterazine3 and 2-deoxycoelenterazine. The designed active sites position an arginine guanidinium group adjacent to an anion that develops during the reaction in a binding pocket with high shape complementarity. For both luciferin substrates, we obtain designed luciferases with high selectivity; the most active of these is a small (13.9 kDa) and thermostable (with a melting temperature higher than 95 °C) enzyme that has a catalytic efficiency on diphenylterazine (kcat/Km = 106 M−1 s−1) comparable to that of native luciferases, but a much higher substrate specificity. The creation of highly active and specific biocatalysts from scratch with broad applications in biomedicine is a key milestone for computational enzyme design, and our approach should enable generation of a wide range of luciferases and other enzymes.
We
report the development of new side-chain amino acid-functionalized
α-helical homopolypeptides that reversibly form coacervate phases
in aqueous media. The designed multifunctional nature of the side-chains
was found to provide a means to actively control coacervation via
mild, biomimetic redox chemistry as well as allow response to physiologically
relevant environmental changes in pH, temperature, and counterions.
These homopolypeptides were found to possess properties that mimic
many of those observed in natural coacervate forming intrinsically
disordered proteins. Despite ordered α-helical conformations
that are thought to disfavor coacervation, molecular dynamics simulations
of a polypeptide model revealed a high degree of side-chain conformational
disorder and hydration around the ordered backbone, which may explain
the ability of these polypeptides to form coacervates. Overall, the
modular design, uniform nature, and ordered chain conformations of
these polypeptides were found to provide a well-defined platform for
deconvolution of molecular elements that influence biopolymer coacervation
and tuning of coacervate properties for downstream applications.
Catalytic
enantioselection usually depends on differences in steric
interactions between prochiral substrates and a chiral catalyst. We
have discovered a carbene Si–H insertion in which the enantioselectivity
depends primarily on the electronic characteristics of the carbene
substrate, and the log(er) values are linearly related to Hammett
parameters. A new class of chiral tetraphosphate dirhodium catalysts
was developed; it shows excellent activity and enantioselectivity
for the insertion of diarylcarbenes into the Si–H bond of silanes.
Computational and mechanistic studies show how the electronic differences
between the two aryls of the carbene lead to differences in energies
of the diastereomeric transition states. This study provides a new
strategy for asymmetric catalysis exploiting the electronic properties
of the substrates.
The influence of testosterone, luteinizing hormone releasing hormone (LHRH) agonist and combinations of these hormones on gonadotropic hormone (GtH) levels in the sexually immature trout was investigated. Both the steroid and releasing hormone preparations, testosterone in Silastic capsules and cholesterol-pelleted LHRH-A, were formulated for sustained release and long-term biological action following a single hormone implantation. Marked increases in pituitary GtH followed testosterone and/or testosterone and LHRH analogue treatment combined, but the low pituitary GtH level in controls remained unchanged after LHRH analogue administration alone. Plasma GtH titers increased with time after testosterone treatment, indicating a positive steroid feedback effect by androgen on GtH in the juvenile rainbow trout. When combined with testosterone treatment, LHRH analogue augmented plasma GtH levels compared to fish receiving testosterone treatment alone. In males the elevated plasma GtH levels were associated with testes stimulation and onset of spermatogenesis; in females, however, no significant stimulation of the ovaries was observed. It can be concluded from these studies that the testosterone stimulus is sufficient to induce onset of sexual development in immature males but not females. Whereas LHRH analogue releases GtH from the testosterone-primed trout pituitary, LHRH treatment alone under these conditions fails to stimulate the juvenile trout reproductive system.
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