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Supplementary Notes
Reference Number: 2005-01-00642B
Methods
Strain Construction, Protein Expression and Purification
A wild-type RNase A clone was a generous gift from Prof. Ronald Raines at the
University of Wisconsin at Madison. The RNase A gene was subcloned into the SalINcoI sites of the pET-32b vector system. The H12A and H119A single mutants were
generated by site-directed mutagenesis. The GQ10G, GQ7G and the G9 sequences were
inserted in the hinge loop region (following Gly112) of the mutant RNase A by twostep insertion PCR. The GNNQQNY expansion was formed by inserting NQQNYGG
following Asn 113 (The GN corresponds to residues 112 and 113 of the wild-type
protein and NQQNYGG corresponds to the inserted sequence.)
pET32-b plasmids containing the RNase A mutants described above were transformed
into the BL21 (DE3) expression system. Cells were grown in LB-Ampicillin to an OD
of 0.6-0.8 at 600nm and induced with 0.2 mM IPTG. The cells were then grown for an
additional 2 hours post-induction and harvested at 5000xg for 10 minutes. The cell
pellets were lysed by sonication in a buffer that contained 50 mM Tris-HCl at pH 8.0,
detergents, protease inhibitors and lysozyme. The lysed cells were spun down, and the
pellets were resuspended in 50mM Tris, pH 8.0 and taken through another round of
sonication. The supernatant was then dialyzed overnight against 20 mM Tris (pH 8.0),
0.5 M NaCl at 4 C.
The dialyzed samples were loaded on a Pharmacia Hi-Trap Ni-chelating column and
eluted with a 0-250 mM imidazole gradient in 20 mM Tris (pH 8.0), 0.5 M NaCl. The
eluted fractions were dialyzed against enterokinase buffer and digested overnight with
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enterokinase. The digested samples were loaded on the Ni-chelating column again. This
time the flow-through was collected, concentrated and dialyzed against water, pH 7.0,
overnight at 4˚C.
Fibril formation
The purified mutants (pH 7.0) were set up for fibril formation by incubating in 50%
acetic acid at concentrations ranging from 0.15-1.5mM. This was followed by freezing
the samples in dry ice and lyophilization overnight. The lyophilized samples were
resuspended in 30-40 l of water and stored on the benchtop.
Activity Assays
The fibril samples were tested for activity by employing a fluorescence assay kit that is
commercially available from Ambion Inc. Three sets of fibril samples were generated
for the activity assays. The first set comprised of fibrils produced from the Q10-H12A
mutant. The second set comprised of fibrils produced from the Q10-H119A mutant. The
third set comprised of fibrils formed by mixing equimolar amounts of Q10-H12A and
the Q10-H119A mutants before lyophilization. The fibrils were separated from smaller
oligomers by running the samples out on a native gel. The fibril bands that stalled in the
stacking gel were excised and soaked overnight in water, pH 7.0. These overnight
solutions were then tested for activity by carrying out the fluorescence assay. Briefly,
the assay employs a fluorophore-quencher pair separated by 5-6 bases. When the RNase
A cleaves at the RNA base, the fluorophore and quencher become separated in space
and the fluorescence of the fluorophore (reported in relative fluorescence units (RFU))
is then a measure of the specific activity of the RNase. The assays were carried out in
duplicates. The negative control was the substrate in the assay buffer. The positive
control was the wild-type RNase A. The rate of formation of the product is determined
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by the slope of the traces. The slope of a trace divided by concentration of the protein
yields the specific activity of each of the samples. The concentration of the solutions
was determined by measuring absorbance at 277.5nm and by the Bradford Assay. The
rate of formation of the product/unit time/mg of the wild-type protein was set at a 100%.
The activities of the mutants were reported relative to the activity of the wild-type
protein. Similar activity assays were carried out for the wild-type RNaseA to verify that
there are no fibrils that stall in the stacking region.
Electron Microscopy
Carbon-coated parlodion films mounted on copper grids were glow-discharged in order
to increase their hydrophilicity. Four l of each fibril sample was applied to the grids
and incubated for 2 minutes. The grids were then washed three times with water and
finally stained with 4 l of a 2% uranyl acetate solution for 45 seconds. The samples
were then analyzed in a Hitachi H-7000 electron microscope at an accelerating voltage
of 75 keV.
Congo Red Assay
The fibrils were incubated in Congo red solution (prepared as described previously1) for
1 hour. The fibrils were then washed twice by spinning down the samples at 15,000xg
for 5 minutes and resuspending the pellet each time in water. Five l of each sample
was placed on a silanized cover slip and allowed to dry. The dried samples were imaged
using both unpolarized and polarized light from a light microscope.
Native (Non-Denaturing) Gels
Samples were loaded on 8-25% native gels and run on the Pharmacia PHAST gel
system. Since the mutants are basic proteins, reverse electrodes and buffer strips were
used as per the Pharmacia product manual. The gels were visualized by coomassie blue
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staining and silver staining. The silver staining was carried out using the Bio-Rad silver
staining kit.
Model Construction
The β-sheet spine was constructed using idealized β-strands corresponding in sequence
to the Q10 extended hinge-loop region of RNase A. The relative positions of the strands
and the spacing between sheets were taken from the crystal structure of GNNQQNY23
using the two strands of the dry interface as a model. The length of the strands in the
spine model is limited by the requirement for domain swapping; a functional globular
domain must lie at either end of each strand of the spine. Thus, residues involved in
forming the active site of RNase A cannot be recruited into the spine, yet the length of
the strands in the spine must be sufficiently long to provide stability to the amyloid and
a spine radius large enough to accommodate the steric bulk of globular domains
anchored around the spine. We found that the number of residues in the spine could be
maximized if each sheet was made antiparallel rather than parallel. The midpoint of the
ten-residue glutamine stretch was used as the center of the spine and glutamine side
chain torsion angles were adjusted to reproduce the steric zipper observed in the
GNNQQNY crystal structure. We found that the steric zipper could be easily
accommodated by either parallel (as in the GNNQQNY crystal structure) or antiparallel
sheets (as depicted in our RNase model). Glutamine side chains of alternate strands in
the antiparallel sheet must have different sets of rotamer values in order to perpetuate
the polar zipper; both sets are favorable. A 7 twist was introduced into successive
rungs of the spine to simulate the twisted appearance of fibrils in the EM. The graphics
program O was used to orient the functional domains around the hinge-loops of the
core. Functional domains were oriented to permit sterically reasonable connections
between functional domain and hinge loop residues. The positions of the functional
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domains were then adjusted to avoid steric clash with neighboring functional domains.
The modeling process was facilitated by the ability to generate and update the positions
of fibril symmetry-related copies in real time as they were being adjusted. Backbone
conformations for the linkages between hinge-loops and functional domains were
selected from a library of low-energy loop conformations when appropriate. This crude
model was then energy minimized using the program CNS with van der Waals,
electrostatic, and hydrogen bonding terms.
Control Experiments for Activity of Mixed Mutant Fibrils (Fig.2, Table S1)
There are two conceivable alternative explanations for the activity attributed to the
mixed mutant amyloid-like fibrils, both of which we have ruled out.
One alternative is that domain-swapped, complementary, small oligomers cling to
the fibrils in a non-specific manner resulting in apparently active fibrils. We control for
this by separating the smaller oligomers from the fibrils on a non-denaturing gel (Fig 3,
right panel). The fibril bands stall at the surface of the stacking gel and display Congo
red birefringence. These bands are extracted from the gel, soaked in buffer and assayed
for RNase activity. The mixed fibril bands show ~ 12% of the wild type RNase A
activity (orange band, orange trace, Fig. 3). In order to test whether residual oligomers
cling to the fibrils, the fibril bands were filtered through a 0.1m filter and the filtrate
was found to exhibit minimal activity above background (brown trace, Fig.3).
Subsequent washing of the filter did not yield additional activity. The possibility that
the RNase A activity of the mixed fibrils is caused by clinging small oligomers was
subjected to another test: The small oligomers formed from each of the single mutants
Q10-H12A and Q10-H119A and from the mixed sample Q10-H12A+ Q10-H119A were
separated from fibrils by filtration and assayed for activity. The mixed small oligomers
exhibited significantly higher activity than each of the single mutant small oligomers,
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because the mixed oligomers can form active functional units by domain swapping
(Table S1 in Supplementary Information). These active small oligomers were then
added to inactive Q10-H12A fibrils and incubated overnight, and then the mixture was
run on a native gel. The fibril band when extracted showed no increase in activity over
the original Q10-H12A fibrils. We conclude that the native gels are effective in
separating fibrils from small oligomers and hence the activity from the mixed fibrils
does not arise from clinging small oligomers.
Another source of activity may be large, non-fibrillar, domain-swapped, active
aggregates in the stalled band. That is, the stalled band may contain two species inactive amyloid-like fibrils and an active domain-swapped aggregate. Arguing against
this, the samples containing fibrils have been examined by electron microscopy and
have been found not to contain any other large aggregates. Further, the appearance of
the stalled band in the native gel correlates exactly to the detection of fibrils by
microscopy. That is, in cases where the fibrils form over several days following
lyophilization, the first appearance of fibrils detected by EM corresponds exactly to the
first appearance of a stalled band on the native gel. Further, when fibrils were run on
Blue-Native gels 2 that are used to separate very large aggregates (to the order of
Megadaltons), the fibrils remain stalled in the stacking gel and no other higher order
aggregates could be detected. In short, we have found no evidence for contamination of
fibrils by non-fibrillar, large, domain-swapped aggregates of RNase A molecules.
References
1. Ivanova, M.I., et al., An amyloid-forming segment of 2-microglobulin suggests a
molecular model for the fibril. Proc Natl Acad Sci USA July 20; 101(29): 10584-10589
(2004).
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2. Schagger, H., Cramer, W.A. & Jagow G.V. Analysis of molecular masses and
oligomeric states of protein complexes by blue native electrophoresis and isolation of
membrane protein complexes by two-dimensional native electrophoresis. Anal
Biochem. Mar; 217(2): 220-30 (1994).
Table S1. RNase A enzymatic activities for various fibrils and small oligomers
demonstrating that complementation of inactive RNase molecules creates
active fibrils.
The bands stalled in the stacking gel excised from lanes 4, 5 and 6 of
supplementary figure S2 have negligible activity compared to the wild-type
protein (sample 2 in green). The activities of the two single mutants oligomers,
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Q10-H12A, Q10-H119A are negligible (sample 6 and 7) and comparable to their
fibrils (sample 3 and 4). The small oligomers from the domain-swapped mixture
Q10-H12A+Q10-H119A (sample 8 in red) have activity comparable to their fibrils
(sample 5). The addition of the active mixed oligomers Q10-H12A +Q10-H119A
(sample 8, in red) to the inactive Q10-H12A fibril sample (sample 3) does not
result in any increased activity of the stacking Q10-H12A fibril bands (compare
rows 3 and 9). This demonstrates that the small active oligomers can be
effectively separated from the fibrils by gel electrophoresis. That is the activity
measured for the mixed fibrils (sample 5), originates from domain-swapping and
is not due to active small clinging oligomers.
Supplementary Figure S1. Cross- diffraction from partially oriented Q10H119A RNase A fibrils.
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The Q10-H119A fibrils were spun down, resuspended in water and partially
aligned between the ends of silanized capillary tubes. The diffraction was
obtained with the partially oriented fibrils parallel to the vertical axis. A distinct
4.8Å reflection corresponding to the separation between the -strands is
somewhat stronger along the meridion than the equator. A more diffuse 11Å
reflection corresponding to the separation between the -sheets appears
somewhat stronger along the equator than the meridion. This pattern
corresponds to the classical cross- x-ray pattern associated with amyloid-like
fibrils.
Supplementary Figure S2. Silver-stained, non-denaturing gel comparing
lyophilized Q10 -expanded RNase A with lyophilized wild-type RNase A.
Left to right: Lanes 1, 2 and 3 contain lyophilized Q10-H119A + Q10-H12A, Q10H119A and Q10-H12A RNase A mutants, respectively. All three samples contain
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fibrils confirmed by the presence of the stalled bands in the stacking regions on
the surface of the gel and verified by electron microscopy. Lanes 4, 5 and 6
contain lyophilized wild-type protein samples at concentrations at which the Q10RNase A mutants form fibrils (0.15-1.5mM). There is no visible stacking of fibrils
nor are fibrils detected by EM. Further, the stacking regions from lanes 4, 5 and
6 were excised (though no stalled band is detectable) and tested for activity.
The bands did not exhibit significant activity compared to the wild-type protein.
(Table S1, sample 2).