The
interior of living cells is a dense and polydisperse suspension
of macromolecules. Such a complex system challenges an understanding
in terms of colloidal suspensions. As a fundamental test we employ
neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins)
in a cell-like environment (cell lysate) with explicit control over
crowding conditions. In combination with Stokesian dynamics simulation,
we address protein diffusion on nanosecond time scales where hydrodynamic
interactions dominate over negligible protein collisions. We successfully
link the experimental results on these complex, flexible molecules
with coarse-grained simulations providing a consistent understanding
by colloid theories. Both experiments and simulations show that tracers
in polydisperse solutions close to the effective particle radius R
eff = ⟨R
i
3⟩1/3 diffuse approximately as if the
suspension was monodisperse. The simulations further show that macromolecules
of sizes R > R
eff (R < R
eff) are slowed more
(less) effectively even at nanosecond time scales, which is highly
relevant for a quantitative understanding of cellular processes.
Coronavirus disease-2019
(COVID-19), a potentially lethal respiratory
illness caused by the coronavirus SARS-CoV-2, emerged in the end of
2019 and has since spread aggressively across the globe. A thorough
understanding of the molecular mechanisms of cellular infection by
coronaviruses is therefore of utmost importance. A critical stage
in infection is the fusion between viral and host membranes. Here,
we present a detailed investigation of the role of selected SARS-CoV-2
Spike fusion peptides, and the influence of calcium and cholesterol,
in this fusion process. Structural information from specular neutron
reflectometry and small angle neutron scattering, complemented by
dynamics information from quasi-elastic and spin–echo neutron
spectroscopy, revealed strikingly different functions encoded in the
Spike fusion domain. Calcium drives the N-terminal of the Spike fusion
domain to fully cross the host plasma membrane. Removing calcium,
however, reorients the peptide back to the lipid leaflet closest to
the virus, leading to significant changes in lipid fluidity and rigidity.
In conjunction with other regions of the fusion domain, which are
also positioned to bridge and dehydrate viral and host membranes,
the molecular events leading to cell entry by SARS-CoV-2 are proposed.
Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.
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