Postdoc : protein unfolding under high pressure

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Pascale Chenevier
Postdoc protein unfolding under high pressure
The Gruner group has developed tools to study
solids and liquids under high pressure with X
rays. Why? Pressure can shift thermodynamic
equilibria, just as temperature does, without
changing the chemical environment of the
molecules of interest.
diamond windows
X ray beam
High pressure chamber
In particular, many proteins are structured and
associated in oligomers at room pressure. High
pressures shift the equilibria towards monomeric
states, then unfolded states.
high pressure
Thanks to X-ray scattering, we can investigate
the size and shape of the protein, and determine
in which state it is.
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Why do we study protein unfolding?
Genes determine the sequence of proteins, also
called their primary structure. It is a 1D
information, well known thanks to gene sequencing
techniques.
3D
?
1D
Proteins fold into a 3D structure also called
their tertiary structure. For many proteins, it
is very ordered and can be investigated by
crystallography. This structure determines the
function of the protein.
How does a 1D genetic information turn into a 3D
functional structure? Or how is the function
determined by the sequence?
Intermediates are partly ordered, so that the
sequence does not describe them fully. They are
not ordered enough to form crystals. How to
investigate them? Fluorescence, absorbance and
scattering studies can describe their global
properties.
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One example unfolding of ?? lactoglobulin

• lactoglobulin is a protein of cows milk. Here we
  pressurize ? lactoglobulin (?LG) at room
  temperature. The X ray beam is widened when
  interacting with the protein (q represents the
  angle, with q0.1Å-1 ? 2º).
• ?LG scattering is different at room pressure
  (1bar) and 1000 atmospheres (1kbar).

intensity
Scattering of proteins is gaussian, which means
that the intensity logarithm should decrease
linearly with q² (left plot). The slope gives
the size of the protein ?LG is unfolded at high
pressure, its size is increased because it is
less compact.
?LG 1 bar 21Å
?LG 1 kbar 45Å