Publications by authors named "D Hilvert"

219 Publications

Analysis of electrostatic coupling throughout the laboratory evolution of a designed retroaldolase.

Protein Sci 2021 May 2. Epub 2021 May 2.

Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA.

The roles of local interactions in the laboratory evolution of a highly active, computationally designed retroaldolase (RA) are examined. Partial Order Optimum Likelihood (POOL) is used to identify catalytically important amino acid interactions in several RA95 enzyme variants. The series RA95.5, RA95.5-5, RA95.5-8, and RA95.5-8F, representing progress along an evolutionary trajectory with increasing activity, is examined. Computed measures of coupling between charged states of residues show that, as evolution proceeds and higher activities are achieved, electrostatic coupling between the biochemically active amino acids and other residues is increased. In silico residue scanning suggests multiple coupling partners for the catalytic lysine K83. The effects of two predicted partners, Y51 and E85, are tested using site-directed mutagenesis and kinetic analysis of the variants Y51F and E85Q. The Y51F variants show decreases in k relative to wild type, with the greatest losses observed for the more evolved constructs; they also exhibit significant decreases in k /K across the series. Only modest decreases in k /K are observed for the E85Q variants with little effect on k . Computed metrics of the degree of coupling between protonation states rise significantly as evolution proceeds and catalytic turnover rate increases. Specifically, the charge state of the catalytic lysine K83 becomes more strongly coupled to those of other amino acids as the enzyme evolves to a better catalyst. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/pro.4099DOI Listing
May 2021

The OP Protein Cage: A Versatile Molecular Delivery Platform.

Chimia (Aarau) 2021 Apr;75(4):323-328

Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland;, Email:

Well-defined containers constructed from multiple protein subunits are a unique class of nanomaterial useful in supramolecular chemistry and biology. These protein cages are widespread in nature, where they are responsible for a diversity of important tasks. As such, producing our own designer protein cages, complete with bespoke functionalities, is a promising avenue to new nanodevices, biotechnology and therapies. Herein, we describe how an artificial, computationally designed protein cage can be rationally engineered using supramolecular intuition to produce new functional capsules. Positive supercharging the interior cavity of this porous protein cage enables the efficient encapsulation of oligonucleotides by electrostatically-driven self-assembly. Moreover, the resulting cargo-loaded cages enter mammalian cells and release their cargo, for example siRNA which modulates gene expression. To expand the cargo scope of this proteinaceous container, a higher level of supramolecular complexity can also be introduced. Encapsulation of anionic surfactants affords protein-scaffolded micelles, which are capable of sequestering poorly water-soluble small molecules within their hydrophobic cores. These hybrid particles stably carry bioactive cargo and deliver it intracellularly, thereby increasing potency. Further development of these genetically-encoded materials is ongoing towards specific applications ranging from cell biology to medicine.
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http://dx.doi.org/10.2533/chimia.2021.323DOI Listing
April 2021

Cell-Specific Delivery Using an Engineered Protein Nanocage.

ACS Chem Biol 2021 Apr 21. Epub 2021 Apr 21.

Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland.

Nanoparticle-based delivery systems have shown great promise for theranostics and bioimaging on the laboratory scale due to favorable pharmacokinetics and biodistribution. In this study, we examine the utility of a cage-forming variant of the protein lumazine synthase, which was previously designed and evolved to encapsulate biomacromolecular cargo. Linking antibody-binding domains to the exterior of the cage enabled binding of targeting immunoglobulins and cell-specific uptake of encapsulated cargo. Protein nanocages displaying antibody-binding domains appear to be less immunogenic than their unmodified counterparts, but they also recruit serum antibodies that can mask the efficacy of the targeting antibody. Our study highlights the strengths and limitations of a common targeting strategy for practical nanoparticle-based delivery applications.
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http://dx.doi.org/10.1021/acschembio.1c00007DOI Listing
April 2021

Noncanonical heme ligands steer carbene transfer reactivity in an artificial metalloenzyme.

Angew Chem Int Ed Engl 2021 Apr 20. Epub 2021 Apr 20.

Eidgenossische Technische Hochschule Zurich, Laboratorium für Organische Chemie, Wolfgang Paulistrasse 10, Hoenggerberg HCI F339, 8093, Zürich, SWITZERLAND.

Changing the primary metal coordination sphere is a powerful strategy for tuning metalloprotein properties. Here we used amber stop codon suppression with engineered pyrrolysyl-tRNA synthetases, including two newly evolved enzymes, to replace the proximal histidine in myoglobin with N δ -methylhistidine, 5-thiazoyl-alanine, 4-thiazoylalanine and 3-(3-thienyl)alanine. In addition to tuning the heme redox potential over a >200 mV range, these noncanonical ligands modulate the protein's carbene transfer activity with ethyl diazoacetate. Variants with increased reduction potential proved superior for cyclopropanation and N-H insertion, whereas variants with reduced E o values gave higher S-H insertion activity. Given the functional importance of histidine in many enzymes, these genetically encoded analogues could be valuable tools for probing mechanism and enabling new chemistries.
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http://dx.doi.org/10.1002/anie.202103437DOI Listing
April 2021

Biosynthetic Functionalization of Nonribosomal Peptides.

J Am Chem Soc 2021 Feb 11;143(7):2736-2740. Epub 2021 Feb 11.

Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland.

Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.
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http://dx.doi.org/10.1021/jacs.1c00925DOI Listing
February 2021