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SYNTHETIC PROTEINS FROM MINIATURISATION AND DE NOVO DESIGN
Luigi Di Costanzo, Dipartimento di Scienze Chimiche, Università di Trieste.
The de novo design of metalloproteins is an important step toward the engineering of novel materials, catalysts,
and biosensors. An artificial metallo protein DF1 has been recently structurally investigated.
(1)
DF1 is a noncovalently associated homodimer of two helix-turn-helix hairpin motifs, constituted by 48 aminoacids
and designed in such a way to form a dimetallic site, with C2 symmetry, similar to the binuclear iron site
found in bacterioferritin, ribonucleotide reductase and methane monooxygenase natural proteins (Fig.1).
Several divalent ions like Fe2+, Mn2+, Co2+, Ni2+ and Zn2+ well fit the geometrical environment of the Glu4His2
binuclear site of DF1 and balance glutamate residue charges. The crystal structure of the zinc derivative
of DF1 shows two side-chains of L13 and L13' block access to dimetallic site.(1) The substitution of leucine
residues (L13) with alanine or glycine residues would lead to the formation of cavity large enough to allow the
access of small molecules to the metal centre. Two di-Mn(II) derivatives of L13A-DF1 and L13G-DF1 variants were
crystallised and structurally characterised by X-ray diffraction experiments at the Elettra Synchrotron.
The structure of di-Mn(II)-L13A-DF1 presents three crystallographically independent dimers in the asymmetric unit
linked by five "external" manganese ions.(2)
A trilobate peak of electron density lies in a depression formed
between the coordinating the glutamic residues of the active site, has been interpreted as a dimethyl sulfoxide
molecule (DMSO). DMSO was used in the dissolving process of peptide. The structure of di-Mn(II)-L13G-DF1
presents four crystallographically independent dimers in the asymmetric unit linked by four "external" manganese
ions (Fig.2).
Figure 1
Figure 2
(3) Despite the structural similarity to that of di-Mn(II)-L13A-DF1, a significant difference was
found in the electron density map around the dimetallic site of di-Mn(II)-L13G-DF1. Each dimer of the four
crystallographically independent dimer has a larger amount of water molecules in the protein core with respect
to that found in the structure of the Ala13 DF1 derivative (Fig.2). Furthermore, the more striking feature found
in this structure is the presence of two different maganese coordination environment. In three of the four dimers
a water molecule bridges the two metal ions, while in the other one, two apical water molecules coordinating the
two manganese ions are in trans position with respect to the hystidine ligands. These structures represent an important
step toward the de novo design of a catalytically active helix bundle. Recently we have undertaken the crystallographic
study of Mimochrome IV, an miniaturised hemeprotein, which structure resemble the active site of citochrome b5.
This molecule (3, 7, 12, 17-tetramethylporphryn-2,
18-N8-e-(Ac-Glu1-Ser2-Gln3-Leu4-His5-Ser6-Asn7-Lys8-Arg9-NH2)-propionammide) is the third generation molecule of
the mimochromes series engeneerized with the aim to increase the solubility of the previous molecular models.
(4).
It is composed by two identical nine-peptide chains covalently linked to the propionic groups of a deuteroporphyrin
IX through the epsilon-amino function of Lys. Each peptide sequence bears a His residue in the central position,
that may act as an axial ligand in the metal derivative. The NMR structure solution of the Co(III)-Mimochrome IV
complex assumes an folded structure with an alpha-helical conformation for the peptide moieties. We obtained single
crystals and a complete data set at 1.25 Å resolution of this complex. Several attempts to solve the
phase problem of the complex mimochrome IV-Co(III) by molecular replacement, starting from the NMR coordinates failed.
Furthermore, it was not possible to interpret the E-maps obtained by direct methods.
The presence of a cobalt atom in the structure has suggested to perform an MAD experiment to solve the phase problem.
Four MAD data sets were used to solve the XRD structure of the L isomer of Co(III)-Mimochrome IV.
The structure consists of two peptide chains in a helix conformation, sandwiching the porphyrin plane with a pseudo
C2 symmetry. Comparison with NMR structure (Fig. 3) shows that the two helices in the XRD structure are antiparallel,
whereas the axes of the helices make an angle of 122° in the NMR one.
This is the first crystal structure of an artificial heme protein of the Mimochrome series and represents the starting point to address the future design directions to reproduce in artificial molecules the subtle mechanisms that control the heme functions, thus ensuring the selectivity of the natural systems.
 NMR Structure
 XRD Structure
Figure 2
1 A. Lombardi, C. M. Summa, S. Geremia, L. Randaccio, V. Pavone, W. F. DeGrado Retrostructural analysis of metalloproteins: Application to the design of a minimal model for diiron proteins Proc. Natl. Ac. Sc., 97, 6298, 2000
2. L. Di Costanzo, H. Wade, S. Geremia, L. Randaccio, W. F. DeGrado, V. Pavone & A. Lombardi Toward the De novo Design of a Catalytically Active Helix-Bundle: a Substrate-Accessible Carboxylate-bridged Dinuclear Metal Center J.Am.Chem.Soc., 51, 12749, 2001
3. S. Geremia, L. Di Costanzo, W. F. DeGrado, A. Lombardi, V. Pavone, & L. Randaccio X-ray studies of artificial metalloproteins with 4-helix bundle motif J. Inorg. Biochem., 86, 232, 2001
4 A. Lombardi, F. Nastri, V. Peptide-Based heme-protein Models Chemical Reviews, 101, 3165, 2001
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