14 research outputs found
Convenient Approach to Polypeptide Copolymers Derived from Native Proteins
A convenient approach for the synthesis of narrowly dispersed
polypeptide
copolymers of defined compositions is presented. The controlled denaturation
of the proteins serum albumin and lysozyme followed by an in situ
stabilization with polyethylene(oxide) chains yields polypeptide side
chain copolymers of precisely defined backbone lengths as well as
the presence of secondary structure elements. Supramolecular architectures
are formed in solution because of the presence of hydrophobic and
hydrophilic amino acids along the polypeptide main chain. Polypeptide
copolymers reported herein reveal excellent solubility and stability
in aqueous media and no significant cytotoxicity at relevant concentrations,
and they can be degraded via proteolysis, which is very attractive
for biomedical applications. This “semi-synthetic chemistry”
approach is based on a novel and convenient concept for producing
synthetic polypeptides from native protein resources, which complements
traditional polypeptide synthesis and expression approaches and offers
great opportunities for the preparation of diverse polypeptides with
unique architectures
Self-Assembly of High Molecular Weight Polypeptide Copolymers Studied via Diffusion Limited Aggregation
The
assembly of high molecular weight polypeptides into complex
architectures exhibiting structural complexity ranging from the nano-
to the mesoscale is of fundamental importance for various protein-related
diseases but also hold great promise for various nano- and biotechnological
applications. Here, the aggregation of partially unfolded high molecular
weight polypeptides into multiscale fractal structures is investigated
by means of diffusion limited aggregation and atomic force microscopy.
The zeta potential, the hydrodynamic radius, and the obtained fractal
morphologies were correlated with the conformation of the polypeptide
backbones as obtained from circular dichroism measurements. The polypeptides
are modified with polyethylene oxide side chains to stabilize the
polypeptides and to normalize intermolecular interactions. The modification
with the hydrophobic thioctic acid alters the folding of the polypeptide
backbone, resulting in a change in solution aggregation and fractal
morphology. We found that a more compact folding results in dense
and highly branched structures, whereas a less compact folded polypeptide
chain yields a more directional assembly. Our results provide first
evidence for the role of compactness of polypeptide folding on aggregation.
Furthermore, the mesoscale-structured biofilms were used to achieve
a hierarchical protein assembly, which is demonstrated by deposition
of Rhodamine-labeled HSA with the preassembled fractal structures.
These results contribute important insights to the fundamental understanding
of the aggregation of high molecular weight polypeptides in general
and provide opportunities to study nanostructure-related effects on
biological systems such as adhesion, proliferation, and the development
of, for example, neuronal cells
pH responsive supramolecular core-shell protein hybrids
<p>PEGylation of proteins remains an integral part of macromolecular therapeutics due to its well-known benign effects and pharmacokinetic enhancement properties. We report herein that PEGylation can be taken to the next level of complexity and dynamic behaviour by introducing highly stable but responsive supramolecular handles. By attaching small boronic acid groups onto proteins and salicylhydroxamate moiety to end-functionalise PEG chains, we demonstrate a comprehensive study on the facile assembly/disassembly of a core-shell protein–polymer architecture using fluorescence and microscale thermophoresis on a macromolecular level. In addition, we demonstrate that both the activity and cellular transfer of functional proteins remained conserved throughout the assembly process thus establishing a rapid and orthogonal strategy towards protein PEGylation.</p
Site-Selective Lysine Modification of Native Proteins and Peptides via Kinetically Controlled Labeling
The site-selective modification of the proteins RNase
A, lysozyme
C, and the peptide hormone somatostatin is presented via a kinetically
controlled labeling approach. A single lysine residue on the surface
of these biomolecules reacts with an activated biotinylation reagent
at mild conditions, physiological pH, and at RT in a high yield of
over 90%. In addition, fast reaction speed, quick and easy purification,
as well as low reaction temperatures are particularly attractive for
labeling sensitive peptides and proteins. Furthermore, the multifunctional
bioorthogonal bioconjugation reagent (<b>19</b>) has been achieved
allowing the site-selective incorporation of a single ethynyl group.
The introduced ethynyl group is accessible for, e.g., click chemistry
as demonstrated by the reaction of RNase A with azidocoumarin. The
approach reported herein is fast, less labor-intensive and minimizes
the risk for protein misfolding. Kinetically controlled labeling offers
a high potential for addressing a broad range of native proteins and
peptides in a site-selective fashion and complements the portfolio
of recombinant techniques or chemoenzymatic approaches
“Tag and Modify” Protein Conjugation with Dynamic Covalent Chemistry
The
development of small protein tags that exhibit bioorthogonality,
bond stability, and reversibility, as well as biocompatibility, holds
great promise for applications in cellular environments enabling controlled
drug delivery or for the construction of dynamic protein complexes
in biological environments. Herein, we report the first application
of dynamic covalent chemistry both for purification and for reversible
assembly of protein conjugates using interactions of boronic acid
with diols and salicylhydroxamates. Incorporation of the boronic acid
(BA) tag was performed in a site-selective fashion by applying disulfide
rebridging strategy. As an example, a model protein enzyme (lysozyme)
was modified with the BA tag and purified using carbohydrate-based
column chromatography. Subsequent dynamic covalent “click-like”
bioconjugation with a salicylhydroxamate modified fluorescent dye
(BODIPY FL) was accomplished while retaining its original enzymatic
activity
Confinement-Controlled Water Engenders Unusually High Electrochemical Capacitance
The electrodynamics
of nanoconfined water have been shown
to change
dramatically compared to bulk water, opening room for safe electrochemical
systems. We demonstrate a nanofluidic “water-only” battery
that exploits anomalously high electrolytic properties of pure water
at firm confinement. The device consists of a membrane electrode assembly
of carbon-based nanomaterials, forming continuously interconnected
water-filled nanochannels between the separator and electrodes. The
efficiency of the cell in the 1–100 nm pore size range shows
a maximum energy density at 3 nm, challenging the region of the current
metal-ion batteries. Our results establish the electrodynamic fundamentals
of nanoconfined water and pave the way for low-cost and inherently
safe energy storage solutions that are much needed in the renewable
energy sector
Spatiotemporally Controlled Photolabeling of Genetically Unmodified Proteins in Live Cells
Selective
labeling of the protein of interest (POI) in genetically
unmodified live cells is crucial for understanding protein functions
and kinetics in their natural habitat. In particular, spatiotemporally
controlled installation of the labels on a POI under light control
without affecting their original activity is in high demand but is
a tremendous challenge. Here, we describe a novel ligand-directed
photoclick strategy for spatiotemporally controlled labeling of endogenous
proteins in live cells. It was realized with a designer labeling reagent
skillfully integrating the photochemistries of 2-nitrophenylpropyloxycarbonyl
and 3-hydroxymethyl-2-naphthol with an affinity ligand. Highly electrophilic ortho-naphthoquinone methide was photochemically released
and underwent a proximity coupling reaction with nucleophilic amino
acid residues on the POI in live cells. With fluorescein as a marker,
this photoclick strategy enables time-resolved labeling of carbonic
anhydrase subtypes localized either on the cell membrane or in the
cytoplasm and a discriminable visualization of their metabolic kinetics.
Given the versatility underlined by facilely tethering other functional
entities (e.g., biotin, a peptide short chain) via acylation or (in
cell) Huisgen cycloaddition, this affinity-driven photoclick chemistry
opens up enormous opportunities for discovering dynamic functions
and mechanistic interrogation of endogenous proteins in live cells
pH Responsive Janus-like Supramolecular Fusion Proteins for Functional Protein Delivery
A facile, noncovalent solid-phase
immobilization platform is described
to assemble Janus-like supramolecular fusion proteins that are responsive
to external stimuli. A chemically postmodified transporter protein,
DHSA, is fused with (imino)biotinylated cargo proteins via an avidin
adaptor with a high degree of spatial control. Notably, the derived
heterofusion proteins are able to cross cellular membranes, dissociate
at acidic pH due to the iminobiotin linker and preserve the enzymatic
activity of the cargo proteins β-galactosidase and the enzymatic
subunit of <i>Clostridium botulinum</i> C2 toxin. The mix-and-match strategy described herein opens
unique opportunities to access macromolecular architectures of high
structural definition and biological activity, thus complementing
protein ligation and recombinant protein expression techniques
Dendronized Albumin Core–Shell Transporters with High Drug Loading Capacity
We describe the synthesis of a core–shell biohybrid
consisting
of a human serum albumin (HSA) core that serves as a reservoir for
lipophilic molecules and a cationized shell region consisting of <b>ethynyl-G2.0</b>-<b>PAMAM</b> or <b>ethynyl-G3.0</b>-<b>PAMAM</b> dendrons. The binding capacity of lipophilic
guests was quantified applying electron paramagnetic resonance (EPR)
spectroscopy, and five to six out of seven pockets were still available
compared with HSA. The attachment of <b>ethynyl-G2.0</b>-<b>PAMAM</b> dendrons to HSA yielded a nontoxic core–shell
macromolecule that was clearly uptaken by A549 human epithelial cells
due to the presence of the dendritic PAMAM shell. Significantly higher
loading of doxorubicin was observed for dendronized <b>G2-DHSA</b> compared with the native protein due to the availability of binding
pockets of the HSA core, and interaction with the dendritic shell.
Dendronized <b>G2-DHSA</b>-doxorubicin displayed significant
cytotoxicity resulting from high drug loading and high stability under
different conditions, thus demonstrating its great potential as a
transporter for drug molecules
Chemoselective Dual Labeling of Native and Recombinant Proteins
The
attachment of two different functionalities in a site-selective
fashion represents a great challenge in protein chemistry. We report
site specific dual functionalizations of peptides and proteins capitalizing
on reactivity differences of cysteines in their free (thiol) and protected,
oxidized (disulfide) forms. The dual functionalization of interleukin
2 and EYFP proceeded with no loss of bioactivity in a stepwise fashion
applying maleimide and disulfide rebridging allyl-sulfone groups.
In order to ensure broader applicability of the functionalization
strategy, a novel, short peptide sequence that introduces a disulfide
bridge was designed and site-selective dual labeling in the presence
of biogenic groups was successfully demonstrated