Mesoporous silicates (MPS) have several
advantages for the immobilization
of enzymes and large organic molecules. They possess well-defined
pores and their surfaces can be functionalized by chemical methods.
In this study, the model protein ribonuclease A (RNase A) was encapsulated
in unmodified amino- and carboxy-functionalized rodlike SBA-15 with
pore widths ranging from 4.0 to 5.8 nm. Differential scanning (DSC)
and pressure perturbation (PPC) calorimetric techniques were employed
to evaluate the stability, hydration, and volumetric properties of
the confined protein. In addition, the influence of the solution pH,
the surface functionalization, and cosolvents on the protein immobilization
and the thermal stability of the immobilized protein are reported.
The extent of stabilization depends strongly on the surface characteristics
of the host, such as the charge density, and on geometric parameters,
i.e., the pore size and pore volume. The addition of the chaotropic
agent urea leads to an increased protein loading. Addition of the
kosmotropic agent glycerol has the opposite effect. The stability
of the protein RNase A confined in all the mesoporous silicates is
drastically enhanced and is of the order of ΔT
m ≈ 30 ± 10 °C regarding the increase
in temperature stability. The highest immobilization capacity, fastest
immobilization rate, and maximum thermal stability was achieved for
the surface-functionalized SBA-15-COOH. The increased temperature
stability is probably not only due to the entropy-driven excluded
volume effect but also due to an increased hydration strength of the
protein within the narrow silica pores, similar to the effects compatible
osmolytes impose on protein hydration and stability. The absence of
an expansivity increase of the confined protein after thermal denaturation
indicates that inside the pores complete unfolding of the protein
is not feasible anymore.
Not only drastic temperature- but also pressure-induced perturbations of membrane organization pose a serious challenge to the biological cell. Although high hydrostatic pressure significantly influences the structural properties and thus functional characteristics of cells, this has not prevented life from invading the high pressure habitats of marine depths where pressures up to the 100 MPa level are encountered. Here, the temperature- and pressure-dependent structure and phase behavior of giant plasma membrane vesicles have been explored in the absence and presence of membrane proteins using a combined spectroscopic and microscopic approach. Demixing into extended liquid-ordered and liquid-disordered domains is observed over a wide range of temperatures and pressures. Only at pressures beyond 200 MPa a physiologically unfavorable all gel-like ordered lipid phase is reached at ambient temperature. This is in fact the pressure range where the membrane-protein function has generally been observed to cease, thereby shedding new light on the possible origin of this observation.
We employed FT-IR and NMR experiments to investigate the influence of a cell-mimicking crowding environment on the structure and dynamics of an elastin-like peptide (ELP) with the sequence GVG(VPGVG)3, which – due to a high number of hydrophobic amino acid side chains – exhibits an inverse temperature transition (ITT). As simplified crowding agent, we used 30 wt% Ficoll. The FT-IR data revealed the well-known broad ITT above ~25°C, as observed by the decrease of the relative population of random coil structures and the concomitant increase of type II β-turns. Interestingly, the addition of Ficoll leads to a destabilizing effect of type II β-turn structures. This is in contrast to the expected excluded-volume effect of the macromolecular crowder, but can be explained by weak interactions of the peptide with the polysaccharide chains of the crowding agent. Further, the crowding agent leads to the onset of a reversal of the folding transition at high temperatures. The full assignment of the ELP allowed for a residue-specific investigation of the dynamic behavior of ELP by NMR. Due to a strong change of microscopic viscosity between native/buffered conditions and crowded conditions, relaxation data remain inconclusive with respect to the observation of an ITT. Hence, no quantitative details in terms of internal conformational changes can be obtained. However, temperature dependent differences in the13C relaxation behavior between core and terminal parts of the peptide indicate temperature induced changes in the internal dynamics with generally higher internal mobility at chain ends: This is in full agreement with FT-IR data. In harmony with the FT-IR analysis, macromolecular crowding does not lead to significant changes in the relaxation behavior.
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