Download Protein structure and functions

Lecture 5:
9 PROTEIN STRUCTURES AND
FUNCTIONS,
9.1 NMR
magnetisation of nuclei that have non-zero
spins. The main contribution is from 1H, but
proteins synthesised with 13C and 15N are
also helpful. Transient time domain signals
are detected and converted by Fourier
transformation into frequencies. Different
nuclei give different frequencies or chemical
shifts (expressed as parts per million, ppm).
Figure 9.1.1
environment of the nucleus. The main factors
are hydrogen bonding and proximity to
aromatic rings. The NH protons are
particularly sensitive.
neighbouring atoms. No J coupling occurs
across the peptide bond, so amino acid
residues appear independent in this respect.
The three-bond coupling depends on the
dihedral angle of the central bond. The NOE
on the other hand is dependent on dipoledipole interactions between different nuclear
spins through space, and in practice are only
felt if the atoms are within 5Å.
Figure 9.1.2
dimension is the frequency of a second radio
frequency pulse. COSY (J-correlated
spectroscopy) has cross-peaks that arise from
hydrogen atoms bonded to the same or
adjacent atoms e.g. NH/CαH. NOESY
measures through space interactions (Figure
9.1.3).
given by the α-helix as a result of the
proximity of NHi/NHi+1, and CβH/NHi+1
but not between CαHi/NHi+1. In β-strands
strong NOEs are between CαHi/NHi+1., but
not between NHi/NHi+1. In β -sheets NOEs
are seen between different strands, whereas in
α-helix they are between residues three apart
in the sequence. Tertiary interactions can be
defined in a similar way. The resolution of the
spectra can be improved by using 2H, 13C
and 15N.
Figure 9.1.3
Figure 9.1.4
upper and lower bounds for particular
distances, defined by NOEs. The result of an
NMR structure analysis is a family of
conformers that are rather similar where there
are sufficient restraints to limit the
conformations but which differ, sometimes
radically, where there are few restraints or
where the polypeptide chain is thermally
disordered.
Figure 9.1.5
resolution X-ray structure, but probably not
more and sometimes much less. Where the
NMR structures are well determined, they
superpose well with the equivalent structure
defined by X-ray analysis, with the exception
of some loop regions. There is some evidence
that there are local distortions from crystal
packing in the X-ray structures.
NMR peaks broaden. This puts an upper limit
on molecular weight of structures defined that
is effectively around 20,000 but which can
probably be pushed towards 40,000.
Ambiguities arise for oligomers in
distinguishing intramolecular and
intermolecular interactions, but this can be
overcome by labelling. NMR has made major
contributions in defining the structures of
domains of large multidomain proteins,
particularly where chain termini and linkers
9.2 Supersecondary structures
structure elements packed around, a usually
hydrophobic core. However from the point of
view of understanding structure, evolution
and function, it is often helpful to consider
their organisation in a hierarchy of
supersecondary structures and domains.
proteinase. These form hairpins, which have
stabilising interactions through van der Waals
packing between sidechains in the case of
helices and through hydrogen bonds between
mainchain groups in b-hairpins. However,
very few such hairpins are very stable in the
absence of further interactions with other
parts of the chain, although they may provide
nuclei in the protein folding process.
to be the more stable and is found in 99% of
proteins. The packing between the a-helices
and b-strands seems to be mediated by the
packing of rows of sidechains - ridges - of
one secondary structure into the grooves of
another. There is evidence that this gives rise
to preferred orientations of one secondary
structure to another, although the great variety
of sidechains involved in forming the ridges
means that the angles are quite variable.
Figure 9.2.1. Hierarchical organisation of
secondary structures into supersecondary
structures, motifs and domains.
further assemble to give a great variety of
motifs. Figure 9.2.1 illustrates the simpler
supersecondary structures and motifs that
occur in proteins; they occur in a hierarchical
organisation.
9.3 Globular Proteins and Domains
binding proteins, such as cytochrome b562.
(Figure 9.3.1). However, this is not the only
way of packing helices as we have seen from
the structure of haemoglobin, and many other
arrangements are adopted, particularly when
large cofactors like the haem or other
elements of secondary structure are involved.
Figure 9.3.1 The structures of four-helix
bundle proteins. Note that both of these
proteins will have metal cofactors between
the helices, but this is not always the case.
known as structural domains; they represent
most of the smallest stable globular proteins,
but can also occur as part of larger,
multidomain proteins.
contains b-hairpin and a b-arch so that the
strands bridge two sheets (see -crystallin
Figure 9.3.2). In fact -crystallin (from the
eye lens) is an example in which two similar
motifs pack together; each domains
comprises two Greek-key motifs related by a
symmetry axis (2-fold rotation). Thus, each
crystallin domain comprises two b-sheets,
each with contributions from each of the
Greek key motifs. The two sheets have
hydrophobic core between them.
Figure 9.3.2. The structure of -crystallin.
structure, with not much possibility of
movements of the strands or sheets relative to
each other. In fact in most Ig domains - those
found in immunoglobulins themselves, as
well as in cell surface molecules like the
IGFR, the two sheets are further stabilised by
an inter-sheet disulphide bond between two
cystines facing each other in the core. These
can of course only form in the oxidising
environment outside the cell. In fact in FGFR
they are occasionally mutated, or made
stacked upon each other. Sometimes the two
sheets become hydrogen bonded together to
form a b-barrel, so that the strands run at a
small angle to the barrel axis and form Hbonds all around.