Revision Lecture

1
Revision Lecture

• Practice of a C72SFM exam question

2
The question is your handout

• Candidates should note that some of the questions
  (Q1, Q3) may take 10-15 minutes to read fully. Do
  not be alarmed by this. A good answer to these
  two questions need not require you to write more
  than a couple of sides of an answer book.

3
Panic! Where do I start?

• Look at each figure
• What does it tell you?
• Write that down!

4
Figure 1 and its interpretation

• Multiple subunits
• Density of band alpha is greater maybe more
  than one copy of alpha for every copy of beta,
  delta, epsilon, theta.
• Theta is lost at pH11
• Peripheral to membrane

5
Data not shown

• After enzymatic treatment of the material in lane
  A with a mixture of deglycosidases the molecules
  masses of only those bands labelled a, ß, ? and d
  show an approximately 20 decrease in apparent
  molecular mass (data not shown).

• Interpretation
• a, ß, g and d are glycosylated proteins.

6
Figure 2.
How should this be interpreted?
7
Figure 2 - interpretation
Graph of time against log molecular weight gives
an approx straight line and allows determination
of molecular weight of nAChR, inverse log of 2.4
265 kDa.
8
Analytical ultracentrifugation data
You are given the equation!
Under reducing conditions, a Svedberg coefficient
(s) of 9.0 x 10-13 sec-1 and a diffusional
coefficient (D) of 3.7 x 10-7 cm/sec-1 were
obtained. Under oxidising conditions the
Svedberg coefficient was 13.7 x 10-13 sec-1 and
the diffusional coefficient to 2.8 x 10-7
cm/sec-1. In both instances, assume the partial
specific volume (v) was 0.730, and R, the gas
constant to be 8.314 x 107
Plug in S and d, and you get 220 kDa under
reducing, and 440 kDa under non-reducing conditio
ns
9
Figure 3 and its interpretation

• Protein has 5-fold symmetry.
• Central pore

10
Figure 4 and its interpretation

• Dimensions are 160-200 Å long by 75-100 Å wide
  (just use a ruler!)
• The rod of density has the same dimensions as an
  a-helix
• conformational changes upon ligand binding are
  transmitted over a substantial distance
• Confirms theta to be peripheral and intracellular

11
Figure 5 and Figure 6.
All you did to get from top to bottom was
add ligand and freeze within 5ms. You know that
the channel has an open state lifetime of a 100
ms. So Fig 6 must represent the open state!
12
Interpretation - I

• that the nAChR stoichiometry is likely to be
  a2bgdq (from Figure 1, lane A)
• that the q subunit is peripherally-attached to
  the membrane (from Figure 1, lane B
• that the membrane bound subunits are glycosylated
  (from the data not shown)
• that the molecular weight of the receptor is ca.
  250 kDa (from gel filtration, Figure 2)
• that the molecular weight of the receptor doubles
  under oxidising conditions implying the existence
  of a disulphide linked dimer (analytical
  ultracentrifugation)
• that the receptor has 5-fold pseudo-symmetry
  (from the data in figure 3)
• that the 5 subunits of the pentamer may have
  similar structures (from the data in Figures 3, 5
  and 6)

13
Interpretation - II

• that each subunit contains a single membrane
  spanning helix visible at 9Å resolution (Figure
  4, 5, 6)
• that the dimensions of the receptor are ca.
  160-200 Å long by 75-100 Å wide (from figure 4)
• that the channel has been imaged under closed and
  open conditions
• that the membrane-spanning helix undergoes a
  substantial change in conformation that opens up
  the ion conduction pore (Figure 5 and 6).
• that the conformational change must have been
  transmitted over a substantial distance (from
  figure 4, binding site marked).

14
perfectly acceptable answer get full marks.

• The nicotinic acetylcholine receptor (nAChR) is a
  multi-subunit membrane spanning protein in which
  one of the subunits is only peripherally
  attached, probably associated with the remainder
  of the protein by ionic interactions (subunit q,
  removed by high pH treatment of electric organ
  membranes, Figure 1). The likely number of each
  subunit in the complex is a2ß?dq, as the a
  subunit band is significantly more intense in
  both gels in Figure 1. All the subunits have
  molecular masses between 40 and 60 kDa, although
  several kD of each subunit is due to
  glycosylation as these sugar chains are removed
  by enzymatic treatment. The data in Figure 2, in
  addition to the ultracentrifuge data, enables one
  to estimate the molecular mass of the complex.
  From a plot of log molecular weight vs. elution
  time the molecular weight of the nAChR, including
  the peripheral subunit, is 250 kDa, which is
  backed up by the sedimentation coefficient of the
  reduced protein (220 kDa, does not include the q
  subunit). The increased sedimentation coefficient
  of the oxidised protein is consistent with a
  disulphide bond formed dimer (molecular weight
  increases from 220 kDa to 440 kDa).
• Electron microscopy of the receptor (Figure 3)
  clearly shows the pentameric architecture of the
  protein, and suggests that the subunits aß? and d
  may have similar folds. Figure 4 shows that the
  protein has dimensions of about 200 x 120
  Angstroms, and that at the resolution of the EM
  data (9 Angstrom) only one kinked membrane
  spanning a-helix can be identified. The
  peripheral subunit is clearly localised to the
  intracellular face, and much of each of the other
  subunits is exposed to the extracellular face.
  The protein can be trapped in the open and closed
  conformational states by the binding of ligand
  and the rapid freezing in the liquid ethane. This
  process shows that the helix undergoes a
  conformational change that opens up the pore for
  ionic flux (Figure 5 and 6). It is interesting to
  note that this conformational change must have
  been transmitted a considerable distance as the
  binding site for ligand (Figure 4) is several
  tens of Angstroms away from the