Biophysical Studies of Collagen VII

1
Biophysical Studies of Collagen VII
Lincoln Muhoro Noel Lazo, PhD. Aaron Herrmann,
Karin Green Department of Biochemistry,
Clark University, Worcester, MA 01610
Biochemistry and Molecular Pharmacology, UMass
Medical School, Worcester, MA 01605

Fig.10 CD results Effect of Peptide Concentration
BACKGROUND Human skin is held together
by the anchoring complex an intricate network of
interconnecting globular proteins that ensure the
adhesion of the epidermis to the underlying
dermis. The complex consists of
keratin-intermediate filaments, hemidesmosomes,
anchoring filaments and anchoring fibrils.
Dystrophic Epidermolysis Bullosa (DEB), a rare
genetic disorder characterized by skin blistering
and tissue separation, is caused by mutations in
the anchoring fibrils. Over 100 mutations, mostly
single glycine substitutions, have been
identified in the gene for collagen VII, the
monomer protein in fibril assembly. The molecular
architecture of anchoring fibrils, details of the
mechanism of its formation, and the
intermolecular forces that contribute to fibril
stability have not been elucidated. A
hypothetical model for the formation of anchoring
fibrils has been proposed (Fig. 1), but
biophysical evidence is lacking. Knowledge of the
three-dimensional structure of collagen VII will
provide insights into fibril assembly and also
lead to the identification of structural defects
that may result from glycine substitutions
associated with DEB. This, in turn, may lead to
the future development of novel gene or protein
therapies for affected persons.
Fig.10 CD plot shows the presence of secondary
structure in INT39. As expected, the a-helical
content did not change with peptide
concentration. This is supported by the shape of
the graph, which suggests that INT39 may be
predominantly random coiled. Compare to Figure 6.
Table 1 The table shows the secondary structure
fractions in different INT39 peptide
concentrations. The figures show that majority of
the peptide is highly unstructured and thus can
forms random coils, which, as the hypothesis
suggested, may play a role in the stability of
the C7 triple helix. See Fig. 3 and 4.
Fig.1 Hypothetical model for the formation of
anchoring fibrils (Burgeson, 1993). Two collagen
VII monomers dimerize in an anti-parallel fashion
to form a central 60-nm overlap. This allows the
alignment of cysteine residues, which then form
disulfide bonds (vertical lines). The NC-2
domains are removed by proteolysis. The dimers
then aggregate laterally to form fibrils. The
fibrils are 20-75 nm in diameter with a
cross-striated banding pattern. Intermolecular
interactions that stabilize the fibrils have not
been defined.
Fig.2 Figure illustrating the organization of
the dermal epidermal anchoring complex and level
of disruption in epidermolysis bullosa subtypes.
Fig. 4 Random coil segments formed by the
eight-residue interruption in one triple helix
(shown in green) can loop around adjacent helices
to help form tight interhelical associations that
may be essential for the mechanical stability of
anchoring fibrils.
Fig. 3 Energy-minimized structures of (a)
(Pro-Hyp-Gly)2-PPG-GPG-LSG-(Pro-Hyp-Gly)3 and (b)
(Pro-Hyp-Gly)2-PPG-PKVSVDEP-GPG-LSG-(Pro-Hyp-Gly)3
. End-on views are also shown on the right.
Assuming that the interruption forms a random
coil, the model shows that the possible role of
39-residue interruption in the structure and/or
folding of the C7 triple helix.
Expected Charge States for Monomer Ions 1
4310 g/mol 2 2156 g/mol 3 1437.0 g/mol 4
1078.5 g/mol 5 863.0 g/mol 6 719.3 g/mol 7
616.7 g/mol 8 539.8 g/mol 9 479.9
g/mol 10 432 g/mol
EXPERIMENT DESIGN AND METHODS
Cleavage sites

• CONCLUSIONS FUTURE EXPERIMENTS
• 1) INT39 is predominantly random coil (60),
  suggesting that it may play a role in the
  stability of the C7 helix. This knowledge is
  useful because it provides insight on how
  interruptions may contribute to fibril stability
  and assembly.
• This information could be used in the development
  of novel therapeutic strategies for EB.
• 2) Limited proteolysis results reveal that
  approximately only 13 amino acids (underlined)
  may be involved in the structure of IN39, giving
  further evidence that INT39 is mostly
  unstructured.
• 3) Future experiments include characterizing
  INT39 using 2D NMR and molecular modeling studies
  using INT39 as the interruption.