Analysis of myoglobin adsorption to Cu(II)-IDA and Ni(II)-IDA functionalized Langmuir monolayers by grazing incidence neutron and X-ray techniques
Kent MS, Yim H, Sasaki DY, Satija S, Majewski J, Gog T
LANGMUIR : 90 (9): 20 (7): 2819-2829 MAR 30 2004
The adsorption of myoglobin to Langmuir monolayers of a metal-chelating lipid
in crystalline phase was studied using neutron and X-ray reflectivity (NR
and XR) and grazing incidence X-ray diffraction (GIXD). In this system, adsorption
is due to the interaction between chelated divalent copper or nickel ions
and the histidine moieties at the outer surface of the protein. The binding
interaction of histidine with the Ni-IDA complex is known to be much weaker
than that with Cu-IDA. Adsorption was examined under conditions of constant
surface area with an initial pressure of 40 mN/m. After similar to12 h little
further change in reflectivity was detected, although the surface pressure
continued to slowly increase. For chelated Cu2+ ions, the adsorbed layer
structure in the final state was examined for bulk myoglobin concentrations
of 0.10 and 10 muM. For the case of 10 muM, the final layer thickness was
similar to43 Angstrom. This corresponds well to the two thicker dimensions
of myoglobin in the native state (44 Angstrom x 44 Angstrom x 25 Angstrom)
and so is consistent with an end-on orientation for this disk-shaped protein
at high packing density. However, the final average volume fraction of amino
acid segments in the layer was 0.55, which is substantially greater than
the value of 0.44 calculated for a completed monolayer from the crystal structure.
This suggests an alternative interpretation based on denaturation. GIXD was
used to follow the effect of protein binding on the crystalline packing of
the lipids and to check for crystallinity within the layer of adsorbed myoglobin.
Despite the strong adsorption of myoglobin, very little change was observed
in the structure of the DSIDA film. There was no direct evidence in the XR
or GIXD for peptide insertion into the lipid tail region. Also, no evidence
for in-plane crystallinity within the adsorbed layer of myoglobin was observed.
For 0.1 muM bulk myoglobin concentration, the average segment volume fraction
was only 0.13 and the layer thickness was less than or equal to25 Angstrom.
Adsorption of myoglobin to DSIDA-loaded with Ni2+ was examined at bulk concentrations
of 10 and 50 muM. At 10 muM myoglobin, the adsorbed amount was comparable
to that obtained for adsorption to Cu2+- loaded DSIDA monolayers at 0.1 muM.
But interestingly, the adsorbed layer thickness was 38 Angstrom, substantially
greater than that obtained at low coverage with Cu-IDA. This indicates that
either there are different preferred orientations for isolated myoglobin
molecules adsorbed to Cu-IDA and Ni-IDA monolayer films or else myoglobin
denatures to a different extent in the two cases. Either interpretation can
be explained by the very different binding energies for individual interactions
in the two cases. At 50 muM myoglobin, the thickness and segement volume
fraction in the adsorbed layer for Ni-IDA were comparable to the values obtained
with Cu-IDA at 10 muM myoglobin.