3 research outputs found
Reversal of the Hofmeister Series: Specific Ion Effects on Peptides
Ion-specific
effects on salting-in and salting-out of proteins,
protein denaturation, as well as enzymatic activity are typically
rationalized in terms of the Hofmeister series. Here, we demonstrate
by means of NMR spectroscopy and molecular dynamics simulations that
the traditional explanation of the Hofmeister ordering of ions in
terms of their bulk hydration properties is inadequate. Using triglycine
as a model system, we show that the Hofmeister series for anions changes
from a direct to a reversed series upon uncapping the N-terminus.
Weakly hydrated anions, such as iodide and thiocyanate, interact with
the peptide bond, while strongly hydrated anions like sulfate are
repelled from it. In contrast, reversed order in interactions of anions
is observed at the positively charged, uncapped N-terminus, and by
analogy, this should also be the case at side chains of positively
charged amino acids. These results demonstrate that the specific chemical
and physical properties of peptides and proteins play a fundamental
role in ion-specific effects. The present study thus provides a molecular
rationalization of Hofmeister ordering for the anions. It also provides
a route for tuning these interactions by titration or mutation of
basic amino acid residues on the protein surface
Two Rapid Catalyst-Free Click Reactions for In Vivo Protein Labeling of Genetically Encoded Strained Alkene/Alkyne Functionalities
Detailed
kinetic analyses of inverse electron-demand DielsāAlder
cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition
reactions were conducted and the reactions were applied for rapid
protein bioconjugation. When reacted with a tetrazine or a diaryl
nitrilimine, strained alkene/alkyne entities including norbornene, <i>trans</i>-cyclooctene, and cyclooctyne displayed rapid kinetics.
To apply these āclickā reactions for site-specific protein
labeling, five tyrosine derivatives that contain a norbornene, <i>trans</i>-cyclooctene, or cyclooctyne entity were genetically
encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA<sub>CUA</sub><sup>Pyl</sup> pair. Proteins
bearing these noncanonical amino acids were successively labeled with
a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro
and in living cells
Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoVā2
Using the regioselective cyanobenzothiazole
condensation reaction
with an N-terminal cysteine and the chloroacetamide reaction with
an internal cysteine, a phage-displayed macrocyclic 12-mer peptide
library was constructed and subsequently validated. Using this library
in combination with iterative selections against two epitopes from
the receptor binding domain (RBD) of the novel severe acute respiratory
syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides
that strongly inhibit the interaction between the Spike RBD and angiotensin-converting
enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified.
The two epitopes were used instead of the Spike RBD to avoid selection
of nonproductive macrocyclic peptides that bind RBD but do not directly
inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2
showed that one macrocyclic peptide is highly potent against viral
reproduction in Vero E6 cells with an EC50 value of 3.1
Ī¼M. The AlphaLISA-detected IC50 value for this macrocyclic
peptide was 0.3 Ī¼M. The current study demonstrates that two
kinetically controlled reactions toward N-terminal and internal cysteines,
respectively, are highly effective in the construction of phage-displayed
macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike
epitopes is a promising methodology in the identification of peptidyl
antivirals