maenas (intertidal zone) and Segonzacia mesatlantica - Station ...

150 Current Protein and Peptide Science, 2008, 9, 150-180
The Structural Analysis of Large Noncovalent Oxygen Binding Proteins by
MALLS and ESI-MS: A Review on Annelid Hexagonal Bilayer Hemoglobin
and Crustacean Hemocyanin
Matthieu Bruneaux 1,2 , Morgane Rousselot 1,2 , Emmanuelle Leize 3 , François H. Lallier 1,2 and
Franck Zal 1,2,*
1 UPMC Univ. Paris 06, UMR 7144, Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, Station Biologique
de Roscoff, 29682, France; 2 CNRS, UMR 7144, Station Biologique de Roscoff, 29682, France; 3 CNRS-ULP, UMR 7177,
Laboratoire de Dynamique et Structure Moléculaire par Spectrométrie de Masse, Institut de Chimie, ISIS, 67083 Strasbourg,
France
Abstract: Understanding the function of macromolecular complexes is related to a precise knowledge of their structure.
These large complexes are often fragile high molecular mass noncovalent multimeric proteins. Classical biochemical
methods for determination of their native mass and subunit composition were used to resolve their quaternary structure,
sometimes leading to different models. Recently, the development of mass spectrometry and multi-angle laser light scattering
(MALLS) has enabled absolute determination of native masses and subunit masses. Electrospray ionization mass
spectrometry (ESI-MS) was used in denaturing and native conditions to probe subunit composition and noncovalent assemblies
masses up to 2.25 MDa. In a complementary way, MALLS provides mass and size estimation in various aqueous
solvents. ESI-MS method can also give insights into post-translational modifications (glycosylation, disulfide bridges …).
By combining native mass and subunit composition data, structural models can be proposed for large edifices such as annelid
extracellular hexagonal bilayer hemoglobins (HBL-Hb) and crustacean hemocyanins (Hc). Association/dissociation
mechanisms, protein-protein interactions, structural diversity among species and environmental adaptations can also be
addressed with these methods. With their absolute mass determination, the very high precision of spectrometry and the
versatile nature of light scattering, ESI-MS and MALLS have provided a wealth of data helping to resolve parts of controversies
for HBL-Hb models and opening access to new fields of investigation in structural diversity and molecular adaptation.
In this review we will focus on annelid HBL-Hb and on crustacean Hc and on the original contributions of ESI-
MS and MALLS in this field.
Keywords: Hemoglobin, hemocyanin, mass spectrometry, light scattering, noncovalent macromolecular complexes, structural
model, quaternary structure.
INTRODUCTION
Many biological functions necessary for cellular life are
performed by multimeric protein complexes. Their constituents
can interact noncovalently through electrostatic forces,
hydrogen bonds, van der Waals forces or hydrophobic forces
or be covalently linked by disulfide bonds. These complexes
are either homo- or hetero-polymers. Each subunit can exhibit
an independent activity without any particular properties
emerging from the association step; on the contrary,
some subunits may depend on the interactions within the
complex to gain full activity. To understand the biochemical
mechanisms undertaken by multimeric proteins, the need to
elucidate the subunits identity and arrangement (quaternary
structure) as well as their biochemical properties arises. Once
able to determine structures and resulting functions, and by
relating them to in vivo physico-chemical parameters and
individual life history and environment, one can expect to
lead an integrated physiological approach and to gain insight
into physiological adaptations at the molecular level.
*Address correspondence to this author at the UPMC Univ. Paris 06, UMR
7144, Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, Station
Biologique de Roscoff, 29682, France; Tel: 0033 (0)298292309; Fax:
0033 (0)298292324; E-mail: zal@sb-roscoff.fr
The study of respiratory adaptations in animals is a pertinent
field for leading such an integrated physiological approach.
Respiration is essential for animal life and many
animal phyla have evolved specialized transport molecules in
their circulating fluid permitting enhanced oxygen delivery
to the demanding tissues. These oxygen binding proteins or
respiratory pigments, so-called because of their various colors
when oxygenated, exhibit high structural diversity among
the different groups [1-4].
Biochemical study of the multimeric respiratory pigments
calls for methods able to resolve quaternary structure
of the molecules and identification of the constituting
subunits, as well as characterization of their interactions.
When investigating these complexes, the ideal analytical
technique must be able to preserve these often fragile interactions.
Methods widely used for determination of the native
mass are sedimentation velocity (SV), sedimentation equilibrium
(SE), scanning transmission electron microscopy
(STEM) and size exclusion chromatography (SEC). More
recently, supramolecular mass spectrometry (MS) and multiangle
laser light scattering (MALLS) were also used to ob-
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