13 research outputs found
A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR
Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-C-13(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (similar to 120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40A degrees, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed
The Intracellular Pharmacokinetics of Terminally Capped Peptides
With significant progress in delivery technologies, peptides
and
peptidomimetics are receiving increasing attention as potential therapeutics
also for intracellular applications. However, analyses of the intracellular
behavior of peptides are a challenge; therefore, knowledge on the
intracellular pharmacokinetics of peptides is limited. So far, most
research has focused on peptide degradation in the context of antigen
processing, rather than on peptide stability. Here, we studied the
structure–activity relationship of peptides with respect to
intracellular residence time and proteolytic breakdown. The peptides
comprised a collection of interaction motifs of SH2 and SH3 domains
with different charge but that were of similar size and carried an
N-terminal fluorescein moiety. First, we show that electroporation
is a highly powerful technique to introduce peptides with different
charge and hydrophobicity in uniform yields. Remarkably, the peptides
differed strongly in retention of intracellular fluorescence with
half-lives ranging from only 1 to more than 10 h. Residence times
were greatly increased for retro-inverso peptides, demonstrating that
rapid loss of fluorescence is a function of peptide degradation rather
than the physicochemical characteristics of the peptide. Differences
in proteolytic sensitivity were further confirmed using fluorescence
correlation spectroscopy as a separation-free analytical technique
to follow degradation in crude cell lysates and also in intact cells.
The results provide a straightforward analytical access to a better
understanding of the principles of peptide stability inside cells
and will therefore greatly assist the development of bioactive peptides