68 research outputs found
Polymeric peptide pigments with sequence-encoded properties
Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence–encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine
Interactions of Intact Unfractionated Heparin with Its Client Proteins Can Be Probed Directly Using Native Electrospray Ionization Mass Spectrometry
Heparin and related members of the
glycosaminoglycan (GAG) family
are highly polyanionic linear saccharides that play important roles
in a variety of physiological processes ranging from blood coagulation
to embryo- and oncogenesis, tissue regeneration, and immune response
regulation. These diverse functions are executed via a variety of
mechanisms, including protein sequestration, activation, and facilitation
of their interactions with cell-surface receptors, but deciphering
the specific molecular mechanisms is frequently impossible due to
the extremely high degree of GAG heterogeneity. As a result, the vast
majority of studies of heparin (or related GAGs) interactions with
its client proteins use synthetically produced heparin mimetics with
defined structure or short heparin fragments. In this work we use
native electrospray ionization mass spectrometry (ESI MS) in combination
with limited charge reduction in the gas phase to obtain meaningful
information on noncovalent complexes formed by intact unfractionated
heparin and antithrombin-III, interaction which is central to preventing
blood clotting. Complexes of different stoichiometries are observed
ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding
stoichiometry, the measurements allow the range of heparin chain lengths
to be obtained for each complex and the contribution of each complex
to the total ionic signal to be calculated. Incorporation of ion mobility
measurements in the experimental workflow allows the total analysis
time to be shortened very significantly and the charge state assignment
for the charge-reduced species to be verified. The possibility to
study interactions of intact unfractionated heparin with a client
protein carried out directly by native ESI MS without the need to
use relatively homogeneous surrogates demonstrated in this work opens
up a host of new exciting opportunities and goes a long way toward
ameliorating the persistent but outdated view of the intractability
of such interactions
A New Approach to Measuring Protein Backbone Protection with High Spatial Resolution Using H/D Exchange and Electron Capture Dissociation
Inadequate
spatial resolution remains one of the most serious limitations
of hydrogen/deuterium exchange-mass spectrometry (HDX-MS), especially
when applied to larger proteins (over 30 kDa). Supplementing proteolytic
fragmentation of the protein in solution with ion dissociation in
the gas phase has been used successfully by several groups to obtain
near-residue level resolution. However, the restrictions imposed by
the LC–MS/MS mode of operation on the data acquisition time
frame makes it difficult in many cases to obtain a signal-to-noise
ratio adequate for reliable assignment of the backbone amide protection
levels at individual residues. This restriction is lifted in the present
work by eliminating the LC separation step from the workflow and taking
advantage of the high resolving power and dynamic range of a Fourier
transform ion cyclotron resonance-mass spectrometer (FTICR-MS). A
residue-level resolution is demonstrated for a peptic fragment of
a 37 kDa recombinant protein (N-lobe of human serum transferrin),
using electron-capture dissociation as an ion fragmentation tool.
The absence of hydrogen scrambling in the gas phase prior to ion dissociation
is verified using redundant HDX-MS data generated by FTICR-MS. The
backbone protection pattern generated by direct HDX-MS/MS is in excellent
agreement with the known crystal structure of the protein but also
provides information on conformational dynamics, which is not available
from the static X-ray structure
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