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The Structural Analysis of Large Noncovalent Oxygen Binding Proteins Current Protein and Peptide Science, 2008, Vol. 9, No. 2 155
fer into a gas phase is pertinent. To obtain reliable masses,
the interactions between subunits must be maintained
throughout the ionization and analysis process. The mechanism
of ESI is not fully understood yet [75]. During ionization
the different kinds of interaction are probably modified
in different ways: hydrophobic interactions are thought to be
broken while hydrogen bonds and electrostatic and van der
Waals interactions could be favored [53]. Successful recovery
of viable viruses after ESI and quadrupole filtering
strongly suggests that native conformation is retained
throughout the ionization process [76]. However, a limit for
the size of analyzed macromolecular assemblies could be the
droplet size in the spray [77]. Recent studies using computer
simulation of solvent evaporation suggested that the conformation
is influenced but the major structural features such as
secondary structures are conserved [78]. It was also suggested
that the use of basic compounds rather than ammonium
acetate in sample preparation could help to reproduce
more closely the solution-phase conformation in the gasphase
since the charge of the protein would be reduced and
would be less likely to influence the structure [79].
MALLS and ESI-MS have proved to be useful techniques
for monitoring interactions between proteins and
ligands, association and dissociation kinetics, protein folding
and enzymatic mechanisms. The complementarity of
MALLS solvent versatility and ESI-MS high mass accuracy
allows new and original approaches for investigation of large
noncovalent protein structures. We have recently been using
this kind of approach in the study of annelid HBL-Hbs and
crustacean Hc.
4. REBUILDING RESPIRATORY PIGMENT
STRUCTURE FROM EXPERIMENTAL DATA: THE
EXAMPLE OF ANNELID HBL-HB AND CRUS-
TACEAN HC
By using a broad spectrum of biophysical and biochemical
techniques and particularly ESI-MS and MALLS, we
were able to provide precise and coherent structural information
on the subunit types and masses, on the assemblies of
subunits, as well as on the mass, subunit composition, and
isoform multiplicity of the native respiratory pigment.
4.1. Building Models for Lumbricus terrestris and Arenicola
marina HBL-Hb from the Masses
The hexagonal bilayer hemoglobins are ~3.6 MDa complexes
of globin chains and non-globin linker chains found
in annelids and related species. To comprehend the quaternary
structure and to conceive models of these giant Hb has
been a challenge over several years, aiming to understand the
structure-function relationships of these hetero-multimeric
complexes. Even though a total agreement has not yet been
attained, a general consensus emerges from these studies. All
HBL-Hb examined so far are built from 12 subassemblies
composed of globin chains (14-17kDa) and linked together
by a complex central structure comprised of linker chains
(24-32 kDa). Major milestones in this field were the recent
reconstructions of LtHb and AmHb from crystallographic
data [80, 81] (Fig. 1).
The knowledge of the accurate molecular mass of the
native HBL-Hb is crucial for the calculation of the number
of polypeptide chains involved in the hetero-multimeric
complex. However, there is appreciable scattering in the
published values for the mass of HBL-Hb obtained with different
techniques and preparation conditions [32]. In most of
the studies related to structural modeling, MALLS has been
used as a quasi absolute method for determining the molecular
mass of macromolecules since it does not require any
“universal” calibration or other a priori hypothesis.
Since 1996, significant efforts have been devoted by several
laboratories to elucidate in greater details the arrangements
between the subunits of HBL-Hb from the structural
point of view. Ten years ago, Brian N. Green was one of the
first to be able to use maximum entropy deconvolution
(MaxEnt) of the raw ESI-MS spectra of several native and
chemically modified annelid [23, 82-84], vestimentiferan
[68, 85] and pogonophoran [86] Hb in order to obtain complete
zero charge spectra of all their constituent subunits and
polypeptide chains and to determine their masses with an
undreamed accuracy of ± 1-3 Da (for subunits ranging from
16 x 10 3 to 50 x 10 3 Da). MaxEnt analysis of ESI-MS spectra
produces semiquantitative relative intensity data: an estimation
of the number of copies of each chain in the whole
molecule can be derived assuming that each chain behave
similarly, i.e., that the ratio K=(peak intensity)/(number of
copies) is the same for each component [63, 71, 87, 88].
In summary, knowing the mass of the HBL-Hb complexes
as obtained by MALLS (Fig. 1c) and the exact masses
of each chain as determined by ESI-MS [22, 23, 83, 89] (Fig.
1e), and further assuming that the molecule possesses a D 6
point-group symmetry, as evidenced by TEM (Fig. 1a), 3D
reconstruction by cryoelectron microscopy [90, 91] (Fig. 1b)
and crystallography in some cases [80, 81] (Fig. 1g), structural
models can be proposed for the HBL-Hbs (Fig. 1f).
The entire HBL-Hb complexes of ~3 600 kDa have not
yet been observed by mass spectrometry even if an unresolved
signal at ~22500 m/z could be attributed to AmHb
HBL-Hb as observed in Fig. (7a). Several reasons can explain
this fact. Firstly, divalent cations are needed to maintain
the quaternary structure but provoke adduct formation
and peak enlargement. The existence of a structural polymorphism
which has been evidenced recently for AmHb (see
below –[92]) in combination with a much lower resolution at
this m/z scale makes the detection harder. However, the noncovalent
subassembly comprising 12 globin chains (204 to
214 kDa) was observed directly by electrospray ionization
time-of-flight mass spectrometry in the native hexagonal
bilayer hemoglobins from several annelids [63]. All the native
HBL-Hb complexes analyzed so far exhibited peaks
with charge distributions from 32+ to 38+ and masses from
204 to 214 kDa (Fig. 1d). These were attributed to dodecameric
globin subassemblies on the basis of correspondence
between the experimental and calculated molecular masses
(Table 1,2).
Several HBL-Hb complexes from representative species
of the three groups of annelids (achaetes, oligochaetes and
polychaetes) were investigated using the complementarity of
MALLS and ESI-MS under denaturing and non-denaturing
conditions (see Table (1)). In this review, we will focus on
the proposed structural model of the most extensively stud-
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