CHAPTER 14
Cavity Supported HPLC of Cis/Trans Isomers
of Proline Containing Peptides Using Cyclodextrins
and Calixarenes
S. MENGEl, S. GEBAUER l and GERD-J. KRAUSS l
1
Introduction
In biological systems the cellular metabolism is decisively controlled by the
dynamics of folding and refolding of peptidic structures. Essential processes of
protein biogenesis such as protein synthesis, and translocation of proteins into
intracellular compartments require the protein to exist temporarily in unfolded
or partially folded conformation (Beissinger and Buchner 1998). Therefore,
information on in vivo and in vitro folding and refolding of proteins is of actual
interest in biochemistry and biotechnology and the knowledge of these processes
is of remarkable importance for the production of biological active recombinant
proteins.
The amino acid proline plays an outstanding role in such processes. Proline
represents a molecular switch in the polypeptide chain, which controls the conformational changes of the prolyl bond and, therefore, the alignment of the polypeptide backbone.
In peptides containing proline in a C-terminal position the free rotation
around the peptide bond is hindered due to the formation of a pyrrolidin ring
system with the prolyl nitrogen (Brandts et al. 1975). The C-N bond is marked by
a partial double bond character leading to energy barriers for the rotation
around the peptidyl-prolyl bond, resulting in two lowest energy arrangements for
the prolyl peptide bond: cis and trans. Cis and trans conformers are defined by
the dihedral angle (() with (() ~ DoC, cis and (() ~ 180°C, trans. They exist in the
aqueous peptide solution in comparable amounts due to comparable thermodynamic stabilities, but the trans conformer is a little energetically favored. In principle, polypeptides with n Xaa-proline bonds can form 2 n bond isomers. Frequently, the dynamic cis/trans isomerization of prolyl bond turned out to be the
rate limiting step in folding/refolding processes of proteins (Brandts et al. 1997,
Lin and Brandts 1987). The interconversion of both energetic arrangements can
be catalyzed by peptidyl-prolyl cis/trans isomerases (PPI'ases), which accelerate
the cis to trans isomerization by lowering the rotation barriers (Lang et al. 1987,
Fischer 1994).
1 Martin-Luther-University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Institute
of Biochemistry, Kurt-Mothes-Str. 3, D-06120 Halle/S., Germany.
Cavity Supported HPLC of Cis/Trans Isomers
of Proline Containing Peptides Using Cyclodextrins
and Calixarenes
S. MENGEl, S. GEBAUER l and GERD-J. KRAUSS l
1
Introduction
In biological systems the cellular metabolism is decisively controlled by the
dynamics of folding and refolding of peptidic structures. Essential processes of
protein biogenesis such as protein synthesis, and translocation of proteins into
intracellular compartments require the protein to exist temporarily in unfolded
or partially folded conformation (Beissinger and Buchner 1998). Therefore,
information on in vivo and in vitro folding and refolding of proteins is of actual
interest in biochemistry and biotechnology and the knowledge of these processes
is of remarkable importance for the production of biological active recombinant
proteins.
The amino acid proline plays an outstanding role in such processes. Proline
represents a molecular switch in the polypeptide chain, which controls the conformational changes of the prolyl bond and, therefore, the alignment of the polypeptide backbone.
In peptides containing proline in a C-terminal position the free rotation
around the peptide bond is hindered due to the formation of a pyrrolidin ring
system with the prolyl nitrogen (Brandts et al. 1975). The C-N bond is marked by
a partial double bond character leading to energy barriers for the rotation
around the peptidyl-prolyl bond, resulting in two lowest energy arrangements for
the prolyl peptide bond: cis and trans. Cis and trans conformers are defined by
the dihedral angle (() with (() ~ DoC, cis and (() ~ 180°C, trans. They exist in the
aqueous peptide solution in comparable amounts due to comparable thermodynamic stabilities, but the trans conformer is a little energetically favored. In principle, polypeptides with n Xaa-proline bonds can form 2 n bond isomers. Frequently, the dynamic cis/trans isomerization of prolyl bond turned out to be the
rate limiting step in folding/refolding processes of proteins (Brandts et al. 1997,
Lin and Brandts 1987). The interconversion of both energetic arrangements can
be catalyzed by peptidyl-prolyl cis/trans isomerases (PPI'ases), which accelerate
the cis to trans isomerization by lowering the rotation barriers (Lang et al. 1987,
Fischer 1994).
1 Martin-Luther-University Halle-Wittenberg, Department of Biochemistry/Biotechnology, Institute
of Biochemistry, Kurt-Mothes-Str. 3, D-06120 Halle/S., Germany.