A FIVE-RESIDUE DELETION ALTERS COMPLETELY THE FOLDING AND INCREASES DRASTICALLY THE STABILITY OF ROP
M. Kokkinidis1, G. Cesareni2, H.-J. Hinz3, Y. Papanikolau4 and M. Vlassi4
1University of Crete, Dept. of Biology
& IMBB/FORTH, PO Box 1527, GR-71110 Heraklion, Crete, Greece
2 Dipt. Di Biologia, Il Universita degli
Studi di Roma, Via C, 00173 Rome, Italy
3 Institut f. Physikalische Chemie der
Westfaelischen Wilhelms-Universitaet, Schlossplatz 4/7, 48161
Muenster, Germany
4 NCSR "Demokritos", Ag. Paraskevi
Attikis, PO Box 60228, GR-15130 Athens, Greece
ROP is a dimeric four-alpha-helical bundle protein with two
subunits of 63 amino acids, each consisting of two antiparallel
helices joined by a tight hairpin bend [1]. The simplicity and
high regularity of its structure make ROP an ideal model system
for an analysis of the sequence-structure relationships of
four-alpha-helical bundles [2]. The sequence of the protein
exhibits a characteristic pattern of hydrophobic and hydrophilic
residues which is organized in heptads. The regular heptad
pattern however, breaks down in the bend region. A regular
pattern can be restored either by the insertion of two bend
residues or by the deletion of five. While the insertion of two
residues causes negligible changes to the wild-type structure of
ROP [3], the deletion mutant shows dramatic changes in its
thermodynamic and structural properties. The protein becomes
hyperthermophilic with a transition temperature of T1/2=99oC.
Furthermore, there is a dramatic change in its tertiary and
quaternary structure: At the tertiary structure level the monomer
changes from an antiparallel pair of helices connected by a
hairpin bend, to a single, long helix. At the quaternary
structure level the protein changes from dimer to tetramer,
although the topology of a four-alpha helical bundle is
conserved. The bundle is formed by the long alpha helices of four
monomers. There is a complete reorganization of the hydrophobic
core in this bundle; compared to the wild-type protein, the
mutant shows a simplified pattern of hydrophobic interactions.
The behaviour of the mutant reveals an interesting aspect of
protein folding, in that the protein sequence codes for a
specific topology (four-alpha-helical bundle) rather than for a
specific structure. Topology is established at the quaternary
structure level by the assembly of a suitable number (two or
four) of subunits and is conserved for the bend mutations tested
so far, while the folding of the monomer may drastically change.