Experiments on Biological Structure

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Experiments on Biological Structure - Optical
Microscopy - Electron Microscopy - X-ray
crystallography - NMR Spectroscopy - Fluorescence
Spectroscopy
45 minutes
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Myosin - Optical Microscopy
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Myosin Electron Micrograph
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Wilhelm Conrad Röntgen (1845-1923)
Nobel prize in Physics 1901 in recognition of the
extraordinary services he has rendered by the
discovery of the remarkable rays subsequently
named after him
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Max von Laue (1879-1960)
Nobel prize in Physics 1914 for his discovery of
the diffraction of X-rays by crystals
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Sir William Henry Bragg (1862-1942), William
Lawrence Bragg (1890-1971)
Nobel prize in Physics 1915 for their services in
the analysis of crystal structure by means of
X-rays
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Francis Harry Compton Crick (1916-), James Dewey
Watson (1928-), Maurice Hugh Frederick Wilkins
(1916-)
Nobel prize in Medicine 1962 for their
discoveries concerning the molecular structure of
nucleic acids and its significance for
information transfer in living material
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Diffraction
Scattering a system of two electrons
s0s1/l
phase difference 2pr.(s-s0) 2pr.S
S2sin?/?
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Diffraction
Scattering by a crystal Bragg's law
diffraction as reflection from crystal planes
path difference 2dsin? For constructive
interference, n? 2dsin?
q
q
d
S 2sinq/l 1/d
If a diffraction pattern fades out at an angle of
2qmax, then dmin l / 2sinqmax This is termed
the resolution of the pattern
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End of Lecture 1
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Diffraction
Scattering a system of two electrons
The two waves have equal amplitudes (because e1
and e2 are identical), but a phase difference of
2pr.S
Ss-s02sinq/l
Total scatter (e1) (e2)
1 exp (2pir.S)
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Diffraction
Scattering by an atom Atomic scattering
factor f(S) ? r(r) exp (2pir.S) exp
(2pi-r.S) d3r ? r(r) cos (2pr.S) d3r The
atomic scattering factor is independent of the
direction of S, but does depend on the length of
S S2sinq/l
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Fibre Diffraction from Insect Flight Muscle - 1967
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The Beginning of Molecular Biology. Francis
Crick and James D Watson

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We wish to suggest a structure for the salt of
deoxyribose nucleic acid (D.N.A.). This structure
has novel features which are of considerable
biological interest. A structure
for nucleic acid has already been proposed by
Pauling and Corey (1). They kindly made their
manuscript available to us in advance
of publication. Their model consists of three
intertwined chains, with the
phosphates near the fibre axis, and the bases on
the outside. In our opinion, this structure is
unsatisfactory for two reasons (1)
We believe that the material which gives the
X-ray diagrams is the salt, not the free acid.
Without the acidic hydrogen atoms it
is not clear what forces would hold the structure
together, especially as the
negatively charged phosphates near the axis will
repel each other. (2) Some of the van der Waals
distances appear to be too small.
Another three-chain structure has
also been suggested by Fraser (in the press). In
his model the phosphates are on the
outside and the bases on the inside, linked
together by hydrogen bonds. This structure as
described is rather ill-defined,
and for this reason we shall not comment on it.
We wish to put forward a radically
different structure for the salt of deoxyribose
nucleic acid. This structure has
two helical chains each coiled round the same
axis (see diagram). We have made the usual
chemical assumptions, namely, that
each chain consists of phosphate diester groups
joining ß-D-deoxyribofuranose
residues with 3',5' linkages. The two chains (but
not their bases) are related by a dyad
perpendicular to the fibre axis.
Both chains follow right- handed helices, but
owing to the dyad the sequences of the atoms in
the two chains run in opposite
directions. Each chain loosely resembles
Furberg's model No. 1 that is, the bases are
on the inside of the helix and the
phosphates on the outside. The configuration of
the sugar and the atoms near it is
close to Furberg's 'standard configuration', the
sugar being roughly perpendicular to the attached
base. There is a residue on each
every 3.4 A. in the z-direction. We have assumed
an angle of 36 between adjacent residues
in the same chain, so that the structure
repeats after 10 residues on each chain, that is,
after 34 A. The distance of a
phosphorus atom from the fibre axis is 10 A. As
the phosphates are on the outside, cations have
easy access to them.
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The model of Pauling and Corey

• consists of three intertwined chains, with
  phosphates near the fibre axis, and the bases on
  the outside
• For Watson and Crick unsatisfactory because of
• Salt gives the X-ray diagram and not the free
  acid
• It is not clear what forces hold the structure
  together, especially as the negativ charged
  phosphates near the axis will repel each other
• Some of the van-der-Waals distance appear to
  small

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The structure is an open one, and its water
content is rather high. At lower water contents
we would expect the bases to tilt so that the
structure could become more compact. The novel
feature of the structure is the manner in which
the two chains are held together by the purine
and pyrimidine bases. The planes of the bases are
perpendicular to the fibre axis. The are joined
together in pairs, a single base from the other
chain, so that the two lie side by side with
identical z-co-ordinates. One of the pair must be
a purine and the other a pyrimidine for bonding
to occur. The hydrogen bonds are made as follows
purine position 1 to pyrimidine position 1
purine position 6 to pyrimidine position 6. If it
is assumed that the bases only occur in the
structure in the most plausible tautomeric forms
(that is, with the keto rather than the enol
configurations) it is found that only specific
pairs of bases can bond together. These pairs are
adenine (purine) with thymine (pyrimidine), and
guanine (purine) with cytosine (pyrimidine). In
other words, if an adenine forms one member of a
pair, on either chain, then on these assumptions
the other member must be thymine similarly for
guanine and cytosine. The sequence of bases on a
single chain does not appear to be restricted in
any way. However, if only specific pairs of bases
can be formed, it follows that if the sequence of
bases on one chain is given, then the sequence on
the other chain is automatically determined.
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It has been found experimentally (3,4) that the
ratio of the amounts of adenine to thymine, and
the ration of guanine to cytosine, are always
very close to unity for deoxyribose nucleic
acid. It is probably impossible to build this
structure with a ribose sugar in place of the
deoxyribose, as the extra oxygen atom would make
too close a van der Waals contact. The previously
published X-ray data (5,6) on deoxyribose nucleic
acid are insufficient for a rigorous test of our
structure. So far as we can tell, it is roughly
compatible with the experimental data, but it
must be regarded as unproved until it has been
checked against more exact results. Some of these
are given in the following communications. We
were not aware of the details of the results
presented there when we devised our structure,
which rests mainly though not entirely on
published experimental data and stereochemical
arguments. It has not escaped our notice that
the specific pairing we have postulated
immediately suggests a possible copying
mechanism for the genetic material. Full details
of the structure, including the conditions
assumed in building it, together with a set of
co-ordinates for the atoms, will be published
elsewhere.
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The model of Watson and Crick

• two helical chains each coiled round the same
  axis
• Right handed helices
• Residue on each chain every 3,4 Å in z direction
• Angle of 36 between the residues of the same
  chain
• Structure repeats after 10 residues, after 34 Å
• Bases are joined in pairs via hydrogen bond
• Pairs adenine (purine) thymine (pyrimidin),
  guanine (purine) cytosine (pyrimidin)

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The model of Watson and Crick
D.N.A. BASE SUGAR PHOSPHATE BASE
SUGAR PHOSPHATE BASE SUGAR PHOSPHATE BAS
E SUGAR PHOSPHATE
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The model of Watson and Crick
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The model of Watson and Crick
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Crystallography
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Crystallography

• Rotating X-ray tube
• Heating of the anode caused by electron beam
  limits the power ? rotating cylinder instead of
  fixed piece of metal

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Diffraction
Scattering by a crystal The scattering of a
crystal is zero, because of the large number of
unit cells and because their scattering vectors
are pointing in different directions.
Laue Conditions a.S h b.S k c.S l h, k, l
are whole numbers either zero, positive or
negative
Argand diagramm
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James Batcheller Sumner (1887-1955) Nobel prize
in Chemistry 1946 for his discovery that enzymes
can be crystallized
John Howard Northrop (1891-1987), Wendell
Meredith Stanley (1904-1971) Nobel prize in
Chemistry 1946 for their preparation of enzymes
and virus proteins in a pure form
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John Desmond Bernal (1901-1971) Dorothy Crowfoot
Hodgkin (1910-1994)
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Crystallography

• The main technique behind
• X-ray source X-ray
  detector
• Synchrotron Single photon counter
• Storage ring photographic film
• Rotating anode tube image plates
• Sealed x-ray tube area detectors

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X-ray crystallography - first purify and
crystallize
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Crystallography - Crystals
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Crystallography - Crystals
Unit cell
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Crystallography - Crystals
A crystal is a three dimensional stack of unit
cells !
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Crystallography - Crystals
Different unit cells
a primitive unit cell a unit cell centered

in the
planes a body-centered unit cell a face-centered
unit cell
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Diffraction
Scattering by a crystal F0 (S) S fj
(S) exp (2pirj.S)
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Diffraction
The Result
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Diffraction
Fourier Transformation
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Phase problem
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• Stamp Collecting.

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• Crystallography of Large Complexes.
• Time-Resolved Crystallography.
• Locating Hydrogen Atoms.

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Time Resolved Crystallography.
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Mouse Prion Protein (PrPc) NMR Structure
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Protein Structure
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The Peptide Backbone Chain
Crosslinking - the disulphide bridge
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Side-Chain Conformation
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Linus Carl Pauling (1901-1994)
Nobel prize in Chemistry 1954 for his research
into the nature of the chemical bond and its
application to the elucidation of the structure
of complex substances
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The Alpha Helix (Pauling Corey, from alpha
keratin)
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Supersecondary Structure
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Tertiary Structure
Single helix Alamethicin - a voltage-gated ion
channel antibiotic
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Helix-turn Helix Rop (RNA-Binding Protein)
Four-Helix Bundle
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Two Greek Keys (gamma crystallin)
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DNA-Binding Alpha Domains.
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Cro repressor
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BETA - BARREL PORIN
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Alpha/Beta HORSHESHOE Placental ribonuclease
inhibitor binds very strongly to any
ribonuclease that leaks into the cytosol.
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Same subunit found more than once
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Quaternary Structure Aggregation of tertiary
domains to form a quaternary structure. Photosynth
etic reaction centre
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Potassium Channel
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Aquaporins
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Strands from different protein chains associate
to form 2o struct.
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Same subunit associates