13 research outputs found
Design of Post-Translationally Modified Peptides by Combining Enzymes from Diverse Pathways
Over the past decade, ribosomally-synthesized and post-translationally modified peptides (RiPPs) have emerged as both therapeutically-relevant and engineerable, two traits previously unobserved together in a natural product class. Their biosynthesis is modular: a precursor peptide recruits enzymes that bind one region of the peptide and modify another. This separation of substrate recognition from catalysis allows modifying enzymes to be both highly specific for their peptide and permissive of diverse sequences at the modification site. After modification, the molecules are chemically diverse, sometimes not appearing peptidic at all, and can exhibit picomolar activity for their biological targets in nature. As medium-sized constrained molecules, they also have exciting applications in drug discovery as protein-protein interaction inhibitors, a modality that is currently out of reach of small molecules and antibodies. Despite the therapeutic potential of these molecules, their development has been hampered by a lack of genetic tools and standardized protocols to express, modify, and engineer peptides. Simple peptide expression in a heterologous host, outside the context of a native pathway, is complicated by peptide degradation and solubility, while existing bulky stabilization tags interfere with analytics. As such, efforts to engineer biosynthesis of new RiPPs have been ad hoc, with no formalization of methods to elucidate enzyme-substrate specificities or engineer multi-enzyme pathways. To address this, I utilize a peptide stabilization tag that is small enough for peptides to be analyzed without tag removal, showing both stabilization of diverse peptides and compatibility with their respective modifying enzymes. I then use the stabilization tag and its established expression/purification pipeline to characterize substrate constraints of 9 enzymes in order to engineer biosynthesis of new-to-nature “hybrid peptides”. Collectively, these standardized expression tools, expression conditions, and engineering principles form an enabling platform for future RiPP discovery and engineering.Ph.D
Conformational Dynamics Accompanying the Proteolytic Degradation of Trimeric Collagen I by Collagenases
Collagenases are the principal enzymes responsible for
the degradation
of collagens during embryonic development, wound healing, and cancer
metastasis. However, the mechanism by which these enzymes disrupt
the highly chemically and structurally stable collagen triple helix
remains incompletely understood. We used a single-molecule magnetic
tweezers assay to characterize the cleavage of heterotrimeric collagen
I by both the human collagenase matrix metalloproteinase-1 (MMP-1)
and collagenase from <i>Clostridium histolyticum</i>. We
observe that the application of 16 pN of force causes an 8-fold increase
in collagen proteolysis rates by MMP-1 but does not affect cleavage
rates by <i>Clostridium</i> collagenase. Quantitative analysis
of these data allows us to infer the structural changes in collagen
associated with proteolytic cleavage by both enzymes. Our data support
a model in which MMP-1 cuts a transient, stretched conformation of
its recognition site. In contrast, our findings suggest that <i>Clostridium</i> collagenase is able to cleave the fully wound
collagen triple helix, accounting for its lack of force sensitivity
and low sequence specificity. We observe that the cleavage of heterotrimeric
collagen is less force sensitive than the proteolysis of a homotrimeric
collagen model peptide, consistent with studies suggesting that the
MMP-1 recognition site in heterotrimeric collagen I is partially unwound
at equilibrium
Selection for constrained peptides that bind to a single target protein
AbstractPeptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards “molecular glues” for therapeutics and diagnostics.</jats:p
DNAplotlib: Programmable Visualization of Genetic Designs and Associated Data
DNAplotlib
(www.dnaplotlib.org) is a computational toolkit for the
programmable visualization of highly customizable, standards-compliant
genetic designs. Functions are provided to aid with both visualization
tasks and to extract and overlay associated experimental data. High-quality
output is produced in the form of vector-based PDFs, rasterized images,
and animated movies. All aspects of the rendering process can be easily
customized or extended by the user to cover new forms of genetic part
or regulation. DNAplotlib supports improved communication of genetic
design information and offers new avenues for static, interactive
and dynamic visualizations that map and explore the links between
the structure and function of genetic parts, devices and systems;
including metabolic pathways and genetic circuits. DNAplotlib is cross-platform
software developed using Python
Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products
Cyclic peptide natural products contain
a variety of conserved,
nonproteinogenic structural elements such as d-amino acids
and amide N-methylation. In addition, many cyclic peptides incorporate
γ-amino acids and other elements derived from polyketide synthases.
We hypothesized that the position and orientation of these extended
backbone elements impact the ADME properties of these hybrid molecules,
especially their ability to cross cell membranes and avoid metabolic
degradation. Here we report the synthesis of cyclic hexapeptide diastereomers
containing γ-amino acids (e.g., statines) and systematically
investigate their structure–permeability relationships. These
compounds were much more water-soluble and, in many cases, were both
more membrane permeable and more stable to liver microsomes than a
similar non-statine-containing derivative. Permeability correlated
well with the extent of intramolecular hydrogen bonding observed in
the solution structures determined in the low-dielectric solvent CDCl<sub>3</sub>, and one compound showed an oral bioavailability of 21% in
rat. Thus, the incorporation of γ-amino acids offers a route
to increase backbone diversity and improve ADME properties in cyclic
peptide scaffolds