Set 1 - Problem
Set
I
 
 BI09
Cell
Biology
...

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Unformatted text preview: Problem
Set
I
 
 BI09
Cell
Biology
 
 Instructions:
 
 
 *
Type
your
answers.

 *
Start
each
part
of
the
problem
set
on
a
new
page.
 *
Write
your
FULL
NAME
on
every
page.

 *
 Problem
 Set
 I
 is
 due
 on
 Tuesday
 April
 5th
 at
 5
 pm.
 A
 cardboard
box
will
be
available
on
the
first
floor
of
Broad
 by
Room
114,
to
turn
in
your
work.
 
 
 
 
 
 
 
 
 
 
 
 Part
I.
Protein
Structure
(20
points)
 
 A.
 (7
 points)
 Explain
 the
 role
 of
 water
 in
 the
 hydrophobic
 effect.
 Discuss
 the
 consequences
for
soluble
and
membrane
proteins.
Make
sure
you
comment
on
the
 thermodynamic
origin
of
the
effect.

 
 B.
 (7
points)
 Just
by
knowing
 the
amino
acid
 sequence
 there
is
a
lot
of
information
 you
can
get
about
 a
protein.
Let’s
say
you
are
looking
for
a
membrane
protein
and
 have
two
candidate
amino
acid
sequences
(which
are
given
in
single‐letter
code):
 
 Sequence
1:
 MTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE

 
 Sequence
2:

 AQITGRPEWIWLALGTALMGLGTLYFLVKGMGVSDPDAKKFYAITTLVPAIAFTMYLSML LGYGLTMVPFGGEQNPIYWARYADWLFTTPLLLLDLALLVDADQGTILALVGAAGIMIGT GLVGALTKVYSYRFVWWAISTAAMLYILYVLFFGFTSKAESMRPEVASTFKVLRNVTVVL WSAYPVVWLIGSEGAGIVPLNIETLLFMVLDVSAKVGFGLILLRSRAIFGEAEAPEPSAGDG AAATSD

 
 Use
the
ExPASy
Proteomics
Server
(http://expasy.org/cgi‐bin/protscale.pl
)
to
find
 the
hydropathy
plots
of
each
protein.
Remember
to
use
the
Kyte‐Doolittle
plot
type.
 Which
 one
 looks
 like
 membrane
 protein?
 Explain
 your
 answer
 and
 remember
 to
 include
the
plots
in
your
answers.

 
 C.
 (6
 points)
 Other
information
that
you
can
get
using
the
amino
acid
sequence
is
 the
 secondary
 structure.
 There
 are
 servers
 that
 can
 approximate
 the
 secondary
 structure
 of
 a
 sequence
 using
 simple
 methods.
 Jpred
 is
 one
 of
 them
 (http://www.compbio.dundee.ac.uk/~www‐jpred/).
 Input
 the
 sequence
 of
 the
 membrane
 protein
 and
 get
 your
 secondary
 structure
 prediction.
 
 Compare
 the
 results
with
the
hydropathy
plot
from
part
B.
What
can
you
say
about
the
structure
 of
the
protein?
How
many
times
does
it
cross
the
membrane?
Make
sure
you
include
 the
 sequence
 and
 the
 prediction
 in
 your
 answer.
 Also
 draw
 a
 rough
 topology
 diagram
for
this
protein
in
the
context
of
the
membrane.

 
 Part
II.
Visualization
(20
points)
 
 A.
 (7
points)
 You
are
interested
in
 studying
 the
protein
aravinase
using
antibodies.

 For
 each
 of
 the
 following
 techniques,
 give
 a
 short
 explanation
 of
 the
 methods
 and
 how
it
can
help
you
understand
aravinase’s
function
inside
the
cell:

 
 1.
Immunoprecipitation
 
 2.
Light
microscopy
(two
techniques)
 
 B.
 (7
 points)
 Proteins
 can
 be
 imaged
 in
 cells
 either
 by
 live‐cell
 imaging
 of
 a
 fluorescent
 protein
 such
 as
 GFP,
 which
 is
 expressed
 as
 a
 fusion
 to
 the
 protein
 of
 interest,
 or
 by
 indirect
 immunofluorescence
 of
 fixed
 cells
 using
 antibodies
 specific
 for
 the
 protein.
 
 What
 are
 some
 benefits
 and
 disadvantages
 to
 using
 fluorescent
 protein
fusions
as
opposed
to
indirect
immunofluorescence?

 
 C.
(6
points)
Explain
why
you
can
see
atomic
detail
with
an
electron
microscope
but
 not
alight
microscope.

 
 Part
III.
Function
(20
points)
 
 A.
(4
points)
Describe
the
active
transport,
passive
transport
and
diffusion.

Be
sure
 to
 comment
 on
 sensitivity
 to
 gradients,
 and
 energy
 usage,
 and
 the
 molecules
 that
 utilize
each
type
of
transport.
 
 B.
(4
points)
 You
are
a
researcher
in
a
lab
that
studies
membrane
proteins.

One
of
 your
colleagues
has
developed
an
assay
to
determine
the
concentration
 of
 molecule
 C
 across
 a
 patch
 of
 membrane.
 Molecule
 C
 is
 a
 small,
 uncharged,
 polar
 molecule.
 Below
is
some
of
the
data
collected
by
your
colleague.From
the
graph
below,
explain
 the
nature
of
transport
of
Molecule
C
across
the
membrane.
 

 Why
 do
 you
 get
 saturation?
 Does
 this
 data
 support
 the
 idea
 that
 Molecule
 C
 has
 affinity
for
a
component
of
the
membrane
system?

If
so,
what
would
you
predict
the
 KD
for
the
interaction
to
be?”


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 C.
 (4
 points)
 We
 are
 going
 to
 focus
 our
 attention
 on
 one
 membrane
 protein:
 Bacteriorhodopsin.
 It
 is
 found
 in
 the
 halophilic
 archea
 Halobacterium
 salinarum.
 This
organism
requires
3M
of
NaCl
because
salts
stabilize
many
of
its
cell
structures.
 They
are
typical
aerobes
and
normally
use
O2
to
metabolize
organic
materials
to
get
 energy
from
them.
But
in
its
salty
environment,
O2
has
very
low
solubility.
At
these
 conditions
 Halobacterium
 salinarum
 cannot
 oxidize
 organic
 compounds
 to
 get
 energy.
 Such
 a
 problem
 can
 be
 circumvented
 to
 some
 extent,
 by
 means
 of
 Bacteriorhodopsin.
Explain
the
function
of
bacteriorhodopsin
as
transporter.

What
 does
it
transport?

Is
it
active
or
passive
transport?

 
 D.
(4
points)

What
would
happen
if
you
put
a
muscle
cell
in
de‐ionized
water?

 
 E.
(4
points)
The
Nernst
equation
is
shown
below:
 
 V=(RT/zF)
*
ln(C0
/Ci)
 
 Where
 V
 is
 the
 electrochemical
 potential,
 R
 is
 the
 ideal
 gas
 constant,
 T
 is
 the
 temperature,
 F
is
the
Faraday
constant,
 z
is
the
valence
 (charge)
of
the
ion,
 C0
is
the
 concentration
of
the
ion
outside
and
Ci
is
the
concentration
of
the
ion
inside.

 What
 conditions
 must
 exist
 for
 a
 V=0?
 Assuming
 a
 cation
 like
 Na+,
 how
 do
 the
 concentrations
 change
 if
 the
 potential
 is
 positive
 or
 negative?
 How
 about
 for
 an
 anion
like
Cl‐?

 
 
 
 ...
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This note was uploaded on 01/12/2012 for the course BI 9 taught by Professor Aravin during the Spring '10 term at Caltech.

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