Protein Structure and Dynamics by NMR in Solution
109
Ca 2 + -bound form were plotted as a function of the protein sequence to determine
which regions of the protein module were implicated in Ca 2 + binding (Fig. 7.5.B).
Significant variations of the chemical shift occurred in two distinct regions (residues 127-130 and lSI-ISS), which correspond to those residues that have been
shown to be Ca 2 + -ligands in the crystal structures of analogous, Ca 2 + -binding
EGF-like modules (see for example Rao et al. 1995). It can thus be concluded that
C1r-EGF binds Ca 2 + in an analogous manner to other known Ca 2 +-binding EGFlike modules.
Unfortunately, the quality of the NOESY spectra was not good enough to recalculate the molecular structures from experimental constraints at a sufficient resolution (low signal to noise ratio certainly due to the increased ionic strength and
to losses of the peptide during sample preparation). However, those NOESY patterns which could be observed in these spectra were also present in the spectra of
the apo form and showed identical relative intensities. No new nOe involving the
residues close to the Ca2+ -binding site could be detected, suggesting that Ca 2 +
binding did not lead to major structural variations. The Ca 2 + -bound form of C1rEGF was then modelled in the presence of a Ca 2 + ion, using the experimental constraints obtained from the apo form. Five new distance constraints were added
between the Ca 2 + ion and the carboxyl or carbonyl groups analogous to the Ca 2 +
ligands in the crystal structure of coagulation factor IX (Rao et al. 1995). The
resulting structural ensemble and the ligation of Ca 2 + are shown in Fig. 7.4.B and
C respectively. It can be seen, that Ca 2 + binding induces a significant ordering of
the N-terminal part of the peptide. However, this observation is based only on
the modelled structures and was not reflected in the experimental nOe data. As
it has been proposed that the N-terminal CUB module contributes to Ca 2 + fixation, it might be that Ca 2 + binding leads to conformational changes in the
module-module interface and/or stabilizes a relative orientation of the two modules. NMR experiments on the CUB-EGF module pair could give further insight
into the structural consequences of Ca 2 + binding. However, such a study may be
precluded by the low solubility of this fragment (Thielens et al. 1998).
4.2
Rotational Tumbling and Backbone Dynamics
of Rhodobacter capsulatus cytochrome C2
The cytochromes c play an important role in the transfer of electrons in a large
variety of both eukaryotic and prokaryotic organisms (Pettigrew and Moore
1987). The class I cytochromes c have been used as a model for investigations of
the relationship between structure and function, in particular to understand the
mechanism of electron transfer. The importance of conformational flexibility at
the interaction surface for the function of the cytochrome c has been recognized
and investigated using molecular dynamics simulations and modeling studies
(Northrup et al. 1988, Wendolski and Matthew 1989, Band et al. 1997), but until
now the backbone dynamics of class I cytochromes c have not been investigated
for cytochromes c of known structure.
We have determined the solution structure, local backbone dynamics and
global hydrodynamics of the cytochrome C2 from Rb. capsulatus (Cordier et al.
109
Ca 2 + -bound form were plotted as a function of the protein sequence to determine
which regions of the protein module were implicated in Ca 2 + binding (Fig. 7.5.B).
Significant variations of the chemical shift occurred in two distinct regions (residues 127-130 and lSI-ISS), which correspond to those residues that have been
shown to be Ca 2 + -ligands in the crystal structures of analogous, Ca 2 + -binding
EGF-like modules (see for example Rao et al. 1995). It can thus be concluded that
C1r-EGF binds Ca 2 + in an analogous manner to other known Ca 2 +-binding EGFlike modules.
Unfortunately, the quality of the NOESY spectra was not good enough to recalculate the molecular structures from experimental constraints at a sufficient resolution (low signal to noise ratio certainly due to the increased ionic strength and
to losses of the peptide during sample preparation). However, those NOESY patterns which could be observed in these spectra were also present in the spectra of
the apo form and showed identical relative intensities. No new nOe involving the
residues close to the Ca2+ -binding site could be detected, suggesting that Ca 2 +
binding did not lead to major structural variations. The Ca 2 + -bound form of C1rEGF was then modelled in the presence of a Ca 2 + ion, using the experimental constraints obtained from the apo form. Five new distance constraints were added
between the Ca 2 + ion and the carboxyl or carbonyl groups analogous to the Ca 2 +
ligands in the crystal structure of coagulation factor IX (Rao et al. 1995). The
resulting structural ensemble and the ligation of Ca 2 + are shown in Fig. 7.4.B and
C respectively. It can be seen, that Ca 2 + binding induces a significant ordering of
the N-terminal part of the peptide. However, this observation is based only on
the modelled structures and was not reflected in the experimental nOe data. As
it has been proposed that the N-terminal CUB module contributes to Ca 2 + fixation, it might be that Ca 2 + binding leads to conformational changes in the
module-module interface and/or stabilizes a relative orientation of the two modules. NMR experiments on the CUB-EGF module pair could give further insight
into the structural consequences of Ca 2 + binding. However, such a study may be
precluded by the low solubility of this fragment (Thielens et al. 1998).
4.2
Rotational Tumbling and Backbone Dynamics
of Rhodobacter capsulatus cytochrome C2
The cytochromes c play an important role in the transfer of electrons in a large
variety of both eukaryotic and prokaryotic organisms (Pettigrew and Moore
1987). The class I cytochromes c have been used as a model for investigations of
the relationship between structure and function, in particular to understand the
mechanism of electron transfer. The importance of conformational flexibility at
the interaction surface for the function of the cytochrome c has been recognized
and investigated using molecular dynamics simulations and modeling studies
(Northrup et al. 1988, Wendolski and Matthew 1989, Band et al. 1997), but until
now the backbone dynamics of class I cytochromes c have not been investigated
for cytochromes c of known structure.
We have determined the solution structure, local backbone dynamics and
global hydrodynamics of the cytochrome C2 from Rb. capsulatus (Cordier et al.