Metalloproteins and Metallopeptides –Natural Metallofoldamers - Metallofoldamers: Supramolecular Architectures from Helicates to Biomimetics - page 4

via a reactive oxygen species mechanism, scavenging of hydrogen peroxide, and in the
assembly of cytochrome c oxidase [11].
The absence of the heme from cytochrome c causes complete destabilization of the
protein due to a decrease of hydrophobic contacts as the heme resides at the hydrophobic
core essential for the folding of the protein, an effect similar to guanidinium chloride
denaturation [12]. The denaturant action is related to its competition with water molecules
in the protein binding, resulting in an unfolding of the protein structure. Simulations of
protein folding have shown that correct folding requires full heme contacts at the folding
transition state in addition to the hydrophobic interactions as a critical “folding nucleus”
[13]. The computational results are in agreement with H-D exchange NMR results, sug-
gesting the initiation of the folding by the terminal helices followed by the 60s helix for
the Met80 loop and b-sheets to build onto. The last helix to form is the 40s loop, conclud-
ing the significant folding role of the heme for providing a hydrophobic core to stabilize
the protein and coordinating to the His and Met residues which results in further lowering
the entropy en route to native folding. The heme center can also complicate the folding
process as other residues or small molecules can compete for heme binding, which causes
what is termed “chemical frustration” [14]. Folding of cytochrome c can therefore be
modulated by choosing solvent conditions that favor one set of heme ligands over others.
No crystal structures of the apo protein could be obtained for this reason [10], indicating
the significance of the heme cofactor in forming and maintaining the folding this natural
metallofoldamer cytochrome c.

-Lactalbumin and its Ca 2 þ Binding and Molten Globule 1.2.2.2

a

Calcium binding can cause significant conformational changes, which in turn may medi-
ate a signaling cascade. The “EF-hand” folding is a major Ca-binding motif composed of
a helix-loop-helix sequence as found in the multifunctional messenger calmodulin and
S100. The assembly of the Ca binding can then be propagated to a protein partner with
which the Ca-binding protein is interacting with. In the S100-type of proteins (which reg-
ulate cell cycles, cell growth, and differentiation), Ca binding influences protein folding to
aid in their dimerization and further interaction with other partner proteins. Likewise, Ca
binds a-lactalbumin at a domain containing a helix-loop-helix bend - close to the EF-
hand domain - dubbed a Ca-binding “elbow” [8].
a-Lactalbumin is a main protein component of milk, which has been the target for
investigation of calcium binding to proteins besides the EF-hand group of proteins and
is used as a model for the study of protein stability. It is the regulatory subunit of lac-
tose synthase for the synthesis of lactose from UDP-galactose and glucose in the lactat-
ing mammary gland. The protein possesses a single strong Ca 2þ -binding site, which
can also bind Mg 2þ ,Mn 2þ ,Na þ ,andK þ , and a few distinct Zn 2þ -binding sites. In
bovine a-lactalbumin, Ca 2þ binds to the “elbow” region (Figure 1.3, lavender) via
three carboxylates of Asp82, Asp87, and Asp88, two carbonyl groups of Lys79 and
Asp84, and two water molecules in a distorted pentagonal bipyramid coordination
sphere with the two carbonyl groups at the axial positions. The binding of cations to
the Ca 2þ site increases the stability of a-lactalbumin against heat and various denatur-
ing agents and proteases, while the binding of Zn 2þ to Ca 2þ -saturated protein decreases
the stability and causes aggregation.