Study Guide
BioChem 330 Course Outline September 20, 2011 Part 1
-
University:
Amherst College -
Course:
BIOL 330 | Biochemical Principles of Life at the Molecular Level Academic year:
2011
-
Views:
93
Pages:
23
Author:
l1psm8ck3r34o
BioChem
330
-‐
Course
Outline
September
20,
2011
• Bio-‐molecular
Structure/Func8on
(I)
– PROTEINS
• Structure
–
–
–
–
Chemistry
of
amino
acid
building
blocks
Primary,
secondary
and
terCary
structure
Protein
folding,
thermodynamics
and
kineCcs
PredicCons
of
protein
folding,
dynamics
• FuncCon
– Binding
….a
tale
of
two
globins
(hemoglobin
and
immunoglobulin)
Outline
for
Today
PredicCons
of
Secondary
Structure
Hierarchy
of
Protein
Structure
TerCary
Structure:Folding
Paradigms
α
HELICAL
PACKING
Coiled
coils,
leR
handed
superhelix
Globin
folds
(50o)
and
Felix
folds
(20o)
β
STRUCTURES
Beta
Barrel
forms
hydrophobic
core
in
TIM
Beta
Club
Sandwich
at
the
Interfaces
of
IgG
Secondary
Structural
PredicCons
Secondary
Structural
PredicCons
, . and denote the average values of the probabilities
that a particular will be α -helical, β-strand, or turn. For simplicity
below, we’ve dropped the bracket around the P.
1. Any segment of six or more residues, with Pα >1.03 and Pα > Pβ
and not including Proline is predicted to be a helix.
2. Any seqment of five or more residues, with Pβ > 1.05 as well as Pα
< Pβ. will be β
-sheet.
3. Examine the sequence for tetra-peptides with
Pα
<
0.9,
Pt
>
Pβ.
These have a good chance of being turns.
Chou and Fasman (1974) Biochemistry 13, 222-245.
Hierarchy
of
Protein
Structure
•
•
•
•
Primary
structure
is
determined
by
sequence
Secondary
structure
is
helix,
sheets,
and
turns
or
loops
TerCary
structure
is
packing
of
secondary
structural
elements
Quaternary
is
assembly
of
monomers
into
dimers,
tetramers,
etc.
Fig 1.1 Branden & Tooz
How
do
Two
Helices
Pack
Together?
• Helix
packing:
– bumps
(knobs)
of
the
R
groups
of
one
helix
fit
into
the
grooves
(holes)
between
R
groups
on
the
adjacent
helix
• First
Type:
Coiled
coils
–
–
–
–
–
large
fibrous
proteins:
keraCn,
fibrinogen,
myosin,
acCn,
tropomyosin
many
transcripCon
factors
with
Leu
zippers
helix
is
more
extended
not
quite
alpha:
3.5
residues/turn;
6.5
Å/turn
2
turns:
7
residues,
13
Å
Helices
are
parallel
and
twist
together
in
a
leR
handed
coiled
coil
(2
or
3)
Supercoil
Turn
Length
140
Å
Figure 3.1 Branden & Tooze Fibrinogen How
do
Two
Helices
Pack
Together?
Coiled
coil
packing
a
and
d
are
hydrophobic
• Heptad
repeat
(7
aa)
• Every
fourth
residue
is
hydrophobic
and
oRen
leu
• right
handed
helices
pack
in
an
almost
parallel
fashion
(N
to
C
termini
aligned)
• 18o
Clt
of
helix
axes
lets
knobs
of
one
fit
into
grooves
in
other
How
do
two
helices
pack
together?
• Through
hydrophobic
interacCons
Helical
Wheels
can
help
us
quan5tate
the
effect
– Amphipathic
or
amphiphillic
helix
• hydrophobic
and
hydrophillic
residues
lie
on
opposite
sides
of
the
wheel
• can
act
as
pores
or
channels
in
the
cell
membrane.
– You
can
use
hydropathies
to
calculate
a
Hydrophobic
Moment
of
a
helix
• Each
R
group
is
assigned
a
vector
poinCng
along
its
projecCon
in
helical
wheel
• Length
of
arrow
is
determined
by
magnitude
of
hydropathy
alpha
helix
has
3.6
residues/turn
and
• neg
hydropathy
flips
arrow
180o
• Vector
sum
gives
you
the
hydrophobic
since
there
is
360o/turn
this
means
moment
100o/residue
• TerCary
Structure:
Helical
Packing
• Two
kinds
of
ridges
are
commonly
used
– +4
ridge
defined
by
every
4th
residue
• i.e.
4,8,12
ridge
&
7,11,15
ridge
• these
ridges
are
25o
off
of
the
helix
axis
– +3
ridge
defined
by
every
3rd
residue
•
1,4,7
ridge
and
3,6,9
ridge
• these
ridges
are
45o
off
axis
• Grooves
are
defined
by
the
valleys
between
the
ridges.
3.11
Branden
&
Tooz
This is the top surface
of helix at left
highlighting +4 ridges..
Again,
the
top
surface
of
leRmost
helix
highlighCng
+3
ridges
TerCary
Structure:
Helical
Packing
50o
packing
•
20o
packing
•
3.12
Branden
&
Tooz
(a)
globin
fold
packing
– Flip
helix
II
to
make
an8parallel
packing
–
+4
ridges
into
+4
grooves,
flip
blue
180o,
rotate
one
25o
to
get
aligned,
another
25o
to
nestle
together,
+50o
rela8ve
orienta8on
(b)
FELIX
packing
– Flip
helix
II
to
make
an8parallel
packing
–
+4
ridges
into
+3
grooves,
this
requires
only
20o
reorienta8on
of
blue
helix.
TerCary
Structure:Folding
Paradigms
• ALPHA
DOMAIN
STRUCTURES
Two
Adjacent
Alpha
Helices
are
Usually
An7parallel
• Four
Helix
Bundle
(FELIX)
is
a
common
domain
structure
in
Alpha
Proteins,
gives
a
hydrophobic
core
where
water
is
excluded.
– helical
packing:
helices
pack
at
nearly
20o
away
from
anCparallel,
+4
ridges
from
one
into
+3
grooves
of
the
other.
Predicted
terCary
structure
Epo
– Structures
of
Epo
protein,
Human
(erythropoieCn)
1EER
x-‐ray;
1BUY
NMR
Growth
Hormone
show
this
feature
Makes red blood cells
Tertiary Structure:Folding Paradigms
ALPHA/BETA STRUCTURES
Beta Barrels are commonly formed in the core of a
globular protein from parallel beta strands
Alpha helices on the outside Tertiary Structure:Folding Paradigms
BETA STRUCTURES
Beta Barrel Assemblies: Antibodies, IgG, are almost
exclusively made up with Beta Barrels The
Folding
Problem
Why
is
protein
solubility
a
miracle?
•Anfinsen
Experiments
w
Rnase
•sequence
dictates
structure
•Thermodynamic
Analysis
•the
hydrophobic
effect
•How
can
folding
happen
quickly,
efficiently,
without
dead-‐end
complexes
which
may
be
deadly?
•molecular
chaperones
Swarthmore Yearbook 1938 PROTEINS
VITAL
STATISTICS
• #
amino
acids:
100
to
10,000
• #
atoms:
1,000
to
100,000
(10
atoms/amino
acid)
• Molar
Mass:
10,000
to
1,000,000
daltons
(~110
g/mole
amino
acid)
• density:
similar
to
that
of
crystalline
hydrocarbon
• compressibility:
equal
to
some
metals
• empty
space:
less
than
3%
volume
water
or
internal
caviCes,
very
few
proteins
with
internal
water
Why
is
protein
solubility
a
miracle?
• A
protein
must
be
soluble:
– In
general,
in
order
for
a
protein
to
perform
its
funcCon,
it
must
be
soluble
in
matrix
in
which
it
is
to
funcCon.
Matrix
might
be
cytoplasm;
polar
hydrophilic
environment
or
membrane;
hydrophobic
environment
• A
protein
must
be
globular:
– A
protein
must
be
large
enough
to
perform
catalyCc,
substrate
binding,
regulatory
or
structural
funcCons.
• Problem:
– What
we
are
trying
to
describe
is
a
polymer
of
incredible
rigidity
so
as
to
have
the
density
of
a
solid;
compressibility
of
a
metal,
yet
be
soluble
in
appropriate
medium
and
be
able
to
bind
substrate,
perform
catalyCc
acts,
discriminate
between
correct
and
incorrect
substrates,
bring
two
substrates
together.
Seems
like
we
need
a
certain
amount
of
plasCcity,
exist
in
H2O
with
no
internal
water
• ................seems
like
an
impossible
task! Protein
Folding
is
Primary
Structure
– Anfinsen
proved
in
elegant
experiments
at
NIH
in
1964
that
all
of
the
informaCon
needed
to
fold
a
protein
into
the
correct
3
dimensional
shape
is
contained
in
the
primary
structure
– RNase
is
a
small
protein
(124
amino
acids)
with
4
disulfide
bonds.
(2zpo)
– Rnase
unfolded
by
exposing
the
protein
to
8
M
Urea
(to
destroy
secondary
structure
by
messing
up
H
bonds)
and
β-mercaptoethanol
(to
reduce
disulfides).
Protein
Folding
is
Guided
by
Primary
Structure
– Next,
reduced
RNase
was
allowed
to
refold
by
gently
dialyzing
away
denaturants,
and
allowing
disulfides
to
naturally
air
oxidize.
– About
1%
of
acCvity
was
regained,
signifying
that
at
least
some
part
of
the
protein
had
spontaneously
refolded.
– Remainder
most
likely
has
scrambled
disulfides,
with
eight
cysteines,
many
many
different
possible
ways
of
coupling.
What
happened?
– Full
acCvity
was
regained
by
adding
a
trace
of
reducing
agent
to
the
mixture
so
that
the
RIGHT
disulfides
could
form,
and
the
naCve
structure
could
be
obtained
with
an
acCve
acCve
site
– Proof
posiCve
that
the
Na8ve
Structure
of
a
Protein
is
Fully
determined
by
the
Primary
Sequence,
and
is
a
thermodynamic
minimum
in
energy.
•
“It
struck
me
recently
that
one
should
really
consider
the
sequence
of
a
protein
molecule
about
to
fold
into
a
precise
geometric
form,
as
a
line
of
melody
wriZen
in
canon
form
and
so
designed
by
Nature
to
fold
back
upon
itself,
creaCng
harmonic
chords
of
interacCon
consistent
with
biological
funcCon.
One
might
carry
the
analogy
further
by
suggesCng
that the kinds of chords formed in a protein with scrambled disulfide
bridges...are dissonant, but that, by giving an opportunity for rearrangement
by the addition of mercaptoethanol, they modulate to give the pleasing
harmonics of the native molecule. Whether or not some conclusion can be
drawn about the greater thermodynamic stability of Mozart's over
Schoenberg's music is something I will leave to the philosophers of the
audience.”
Anfinsen (1964) What
factors
stabilize
the
folded
state?
• Disulfide
Bonds
(100s
of
kJ/mole)
• Cofactors
(ATP,
NADH)
or
ProstheCc
groups
(heme)
–
folds
for
these
groups
must
be
there
iniCally
–
flavins,
metals,
sugars,
nucleoCdes,
• Non-‐covalent
InteracCons:
Small
energies
(<10
kJ/mole)
but
many
of
them.
These
small
energies,
are
of
primary
importance.
Right
disulfides
cannot
form
unless
the
correct
structure
is
obtained.
–
–
–
–
–
solvent
interacCons
internal
H-‐bonds
(mostly
-‐C=O…..H-‐N-‐)
salt
bridges
(ion-‐ion
interacCons)
Van
der
Waals
interacCon
CaCon-‐
Pi
InteracCons
• Current
Folding
Paradigm:
A.A.
sequence
specifies
conforma8on,
because
proteins
fold
spontaneously
in
vitro
Are
proteins
rigid
or
flexible?
•
Thermal
B
Factors
in
Crystal
Structure:
Temperature
Factors
lead
to
a
lack
of
resoluCon
in
the
diffracCon
of
x-‐rays
– Substrate
Induced
Changes:
• Hexokinase:
Crystals
grown
in
absence
of
substrate
(glucose)
shaZer
when
substrate
is
added.
• Myoglobin:
Oxygen
can
be
hit
with
light
from
a
laser
which
causes
it
to
photolyze
from
the
molecule,
O2
dissociates,
crystals
shaZer.
Molecular
Dynamics:
Atoms
in
a
protein
can
move,
helices
can
flex,
stretch
and
twist.
AcCvaCon
barriers
to
these
moCons
must
be
within
kT
and
mulCple
energeCc
minima
may
be
found
– MarCn
Karplus,
David
Beveridge
pioneered
ways
to
calculate
posiCons
from
first
principles.
Their
protein
model
system
was
lysozyme
(protein
that
binds
to
polysaccharide
and
cleaves
linkage
sugars).
– Today:
Gerstein
Lab
at
Yale
pioneered
ways
to
visualize
using
morphs:
•
Other
Measurements:
NMR
(see
RCSB),
Fluorescence
• What
is
the
5me
scale
for
protein
dynamics?
• PlasCcity
of
proteins
plays
a
role
in
regulaCon:
Access
to
binding
site.
Release
of
products.
•
•
•
•
•
•
Table
6.4
pg
197
MoCons
in
Protein
Molecules
distance
VibraCons
0.2
Å
Concerted
MoCon
0.2
-‐1.0
MoCons
of
Whole
domains
1-‐10
opening
and
closing
of
cleRs
100
• Examples
and
how
do
you
study?
Proteins 2nd Edition T.E.Creighton
Cme
fs-‐ps
ps-‐10ns
>
10ps
us
BioChem 330 Course Outline September 20, 2011 Part 1
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