1 research outputs found
Site of Tagging Influences the Ochratoxin Recognition by Peptide NFO4: A Molecular Dynamics Study
Molecular
recognition by synthetic peptides is growing in importance
in the design of biosensing elements used in the detection and monitoring
of a wide variety of hapten bioanlaytes. Conferring specificity via
bioimmobilization and subsequent recovery and purification of such
sensing elements are aided by the use of affinity tags. However, the
tag and its site of placement can potentially compromise the hapten
recognition capabilities of the peptide, necessitating a detailed
experimental characterization and optimization of the tagged molecular
recognition entity. The objective of this study was to assess the
impact of site-specific tags on a native peptide’s fold and
hapten recognition capabilities using an advanced molecular dynamics
(MD) simulation approach involving bias-exchange metadynamics and
Markov State Models. The in-solution binding preferences of affinity
tagged NFO4 (VYMNRKYYKCCK) to chlorinated (OTA) and non-chlorinated
(OTB) analogues of ochratoxin were evaluated by appending hexa-histidine
tags (6× His-tag) to the peptide’s N-terminus (NterNFO4)
or C-terminus (CterNFO4), respectively. The untagged NFO4 (NFO4),
previously shown to bind with high affinity and selectivity to OTA,
served as the control. Results indicate that the addition of site-specific
6× His-tags altered the peptide’s native fold and the
ochratoxin binding mechanism, with the influence of site-specific
affinity tags being most evident on the peptide’s interaction
with OTA. The tags at the N-terminus of NFO4 preserved the native
fold and actively contributed to the nonbonded interactions with OTA.
In contrast, the tags at the C-terminus of NFO4 altered the native
fold and were agnostic in its nonbonded interactions with OTA. The
tags also increased the penalty associated with solvating the peptide–OTA
complex. Interestingly, the tags did not significantly influence the
nonbonded interactions or the penalty associated with solvating the
peptide–OTB complex. Overall, the combined contributions of
nonbonded interaction and solvation penalty were responsible for the
retention of the native hapten recognition capabilities in NterNFO4
and compromised native recognition capabilities in CterNFO4. Advanced
MD approaches can thus provide structural and energetic insights critical
to evaluate the impact of site-specific tags and may aid in the selection
and optimization of the binding preferences of a specific biosensing
element