Lecture12-ProBinding

Lecture12-ProBinding - BIOC*2580
Lecture
12.


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Unformatted text preview: BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 1
 
 Protein
Stability,
Binding
and
Recognition

 
 Synopsis:
 The
 purpose
 of
 many
 proteins
 is
 to
 bind
 and
 recognize
 specific
 target
 molecules.
 Such
 proteins
 may
 include
 enzymes,
 which
 catalyze
 a
 reaction
 on
 their
 bound
 target,
 or
 antibodies
that
simply
bind
and
tag
foreign
molecules
for
intervention
by
the
body's
defenses.
 Effective
binding
occurs
through
interaction
very
similar
to
the
interactions
that
cause
proteins
 to
 fold
 up,
 namely
 hydrophobic
 effects,
 van
 der
 Waals
 interactions
 (dependent
 of
 exact
 matching
 of
 molecular
 shape)
 and
 by
 pairing
 up
 opposite
 charges
 or
 hydrogen
 bonding
 partners
 on
 protein
 and
 bound
 ligand.
 Many
 proteins
 are
 able
 to
 bind
 and
 recognize
 specific
 molecules.
 Specific
 binding
 results
 from
 cavities
 and
 regions
 on
 the
 protein
 surface
 which
 complement
the
target
molecule:

 
 non‐polar
 patches
 on
 the
 protein
 surface
 match
 with
 nonpolar
 groups
 on
 the
 ligand
 the
 binding
site
matches
shape
of
ligand
to
maximize
atom
to
atom
contact
oppositely
charged
 groups
on
protein
and
ligand
attract
 complementary
hydrogen
bonding
groups
on
protein
 and
ligand.
 
 Reading:
Lehninger

p.
183‐194
(4th
ed
p.193‐199).
 
 
 Proteins
exist
as
long
polypeptide
chains
folded
into
a
specific
3‐dimensional
 shape.
 
 The
normal
folded
state
or
tertiary
structure
of
a
protein
is
called
its
 native
state.

 The
exact
 spatial
relationship
of
the
amino
acids
in
the
native
state
give
the
protein
a
specific
function.

 
 When
the
polypeptide
 is
 unfolded
from
its
normal
tertiary
structure,
the
protein
is
said
to
be
 denatured.
 
 The
 spatial
 relationship
 of
 amino
 acids
 is
 disorganized,
 so
 a
 denatured
 protein
 loses
 all
 of
 its
 functions.
 
 In
 some
 cases,
 the
 denatured
 state
 may
 exist
 as
 a
 long
 extended
 polypeptide
chain
(e.g.
by
the
action
of
sodium
dodecyl
sulfate
in
SDS
electrophoresis),
but
any
 shape
other
than
the
precise
native
state
is
denatured
and
effectively
inactive.
 
 Covalent
bonding
determines
the
first
level
of
protein
structure.
 
 The
 polypeptide
 backbone
 consists
 of
 a
 chain
 of
 covalently
 bonded
 amino
 acids
 linked
 in
 a
 specific
sequence
‐
the
primary
structure.
 
 Non‐covalent
interactions
give
rise
to
secondary
and
tertiary
structure
and
dictate
the
precise
 pattern
of
folding
and
stability
of
the
folded
form.
 
 These
effects
include
 hydrophobic
interaction,
van
der
Waals
forces,
hydrogen
bonding
and
 ionic
interactions.
 Page
1
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 2
 
 Hydrophobic
effect:
 This
describes
the
tendency
of
non‐polar
amino
acids
to
cluster
in
the
core
of
a
protein,
out
of
 contact
 with
 the
 surrounding
 H2O
 environment.
 This
 effect
 provides
 the
 major
 contribution,
 about
50%
of
the
total,
to
the
energy
that
stabilizes
the
folded
state
of
the
protein.

 
 The
 energy
 actually
 arises
 from
 strong
 interactions
 between
 polar
 amino
 acids
 and
 the
 surrounding
H2O.
When
 nonpolar
amino
acids
concentrate
in
the
 interior,
this
maximizes
the
 number
 of
 polar
 amino
 acids
 on
 the
 exterior,
 where
 they
 interact
 strongly
 with
 the
 surrounding
 H2O.
 Direct
 contact
 with
 nonpolar
 amino
 acids
 has
 energetically
 unfavourable
 effects
on
the
H2O
organization.

Lehninger

p.
47‐49
(4th
ed
p.
52‐54).
 
 The
magnitude
of
the
hydrophobic
effect
can
be
roughly
estimated
by
counting
how
many
CH3‐ ,
 ‐CH2‐
 and
 >CH‐
 groups
 are
 moved
 out
 from
 direct
 contact
 with
 the
 surrounding
 H2O;
 each
 group
removed
contributes
‐5
kJ/mol
to
the
overall
stability.

 
 The
van
der
Waals
effect
 Van
 der
 Waals
 forces
 are
 weak
 electrostatic
 attractions
 between
 atoms
 which
 are
 not
 covalently
 bonded,
 but
 which
 are
 in
 close
 physical
 contact.
 The
 interaction
 arises
 because
 of
 random
 fluctuations
 in
 relative
 distribution
 of
 electrons
 around
 a
 nucleus
 creates
 a
 transient
 dipole.
 In
 turn
 this
 can
 induce
 a
 near
 neighbour
 to
 become
 polarised
 also,
 so
 they
 attract
 each
 other.
 The
net
effect
is
weak
because
the
polarisation
is
temporary.

This
 weak
attractive
force
is
called
a
London
dispersion
force.
 
 Plot
of
the
energy
of
van
der
Waals
interaction
as
a
function
of
distance
 
 When
non‐bonded
atoms
are
too
close
(A)
there
 is
 a
 strong
 repulsion
 indicated
 by
 the
 steep
 negative
 slope
 and
 positive
 free
 energy.
 (B)
 represents
 the
 optimum
 separation
 distance,
 about
 3.4
 Å
 for
 a
 pair
 of
 C
 atoms.
 
 (C)
 A
 moderate
 attractive
 force
 (intermediate
 negative
 energy)
 exists
 provided
 the
 distance
 is
 no
more
than
2‐3
atom
diameters,
otherwise
the
 attractive
 force
 force
 fades
 rapidly
 to
 zero
 (D,
 small
negative
energy).
Although
a
single
van
der
 Waals
 
 interaction
 is
 very
 weak,
 van
 der
 Waals
 forces
 are
 significant
 in
 protein
 because
 thousands
 of
 atoms
 may
 be
 in
 close
 contact
 if
 sidechains
 in
 one
 region
 are
 correctly
 interlocked
 with
 a
 neighbouring
 region.
 
 If
 the
 two
 regions
 don’t
 fit
 each
 other,
 only
 a
 few
 atoms
come
into
direct
contact,
and
some
atoms
may
be
forced
so
close
 that
they
repel
each
 other.

Lehninger

p.
49‐51
(4th
ed
p.54‐55).
 Page
2
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 3
 
 Polar
interactions
may
also
help
stabilize
the
folded
structure
of
a
protein.
 
 Hydrogen
bonds
in
the
polypeptide
backbone
are
important
for
secondary
 structures
 a‐helix
 and
 b‐sheet.
 Some
 hydrogen
 bonds
 may
 form
 between
 adjacent
sidechains
in
the
tertiary
structure.
 
 Ion
pairs
(sometimes
called
salt
bridges)
are
electrostatic
interactions
that
 result
 when
 a
 positive
 group,
 e.g.
 Lys
 or
 Arg
 come
 in
 close
 proximity
 to
 a
 negative,
e.g.
Asp
or
Glu.
 
 Ion
pairs
and
H‐bonds
make
less
contribution
than
hydrophobic
or
van
der
 Waals
 interactions,
 because
 most
 polar
 groups
 face
 the
 surrounding
 aqueous
 medium.

The
majority
of
charged
side
chains
simply
pair
up
with
 solution
 ions,
 e.g.
 K+
 or
 Cl‐,
 or
 with
 dipolar
 water
 molecules
 rather
 than
 linking
with
 other
side
chains.

The
same
applies
to
H‐bonding
side
chains
 since
 H2O
 is
 an
 excellent
 H‐bonding
 agent.
 Peptide
 backbone
 H‐bonds
 involved
 in
 secondary
 structure
 generally
 need
 to
 be
 protected
 from
 the
 surrounding
H2O
by
the
side
chains
of
their
constituent
amino
acids.

 
 Ionic
 and
 H‐bonding
 groups
 may
 act
 more
 to
 destabilize
 wrongly
 folded
 states
than
to
stabilize
the
correctly
folded
state.
 
 In
the
denatured
state
(unfolded):
charged
groups
stabilize
by
hydration.
 In
the
correctly
folded
state:
charged
groups
stabilize
by
forming
opposite
 pairs
or
remain
hydrated.
 In
 any
 wrongly
 folded
 state
 :
 charged
 groups
 that
 become
 unpaired,
 dehydrated
 or
 wrongly
 paired
(+ve
with
+ve
etc)
will
decrease
the
stability
of
the
misfolded
form.
 
 Disulfide
bonds:

covalent
contribution
to
the
tertiary
structure
of
some
proteins
 
 Disulfide
 bonds
 may
 form
 between
 pairs
 of
 cysteines
(‐SH
side
chain),
which
are
physically
close
 in
 the
 folded
 protein.
 Hydrogen
 is
 removed
 from
 paired
 –SH
 groups
 by
 reaction
 with
 O2.
 
 Since
 disulfides
 require
 oxidizing
 conditions,
 whereas
 conditions
 inside
 normal
 cells
 are
 often
 reducing,
 disulfides
 are
 less
 common
 than
 some
 textbooks
 may
 suggest.
 Disulfide
 bonds
 are
 mostly
 limited
 to
 proteins
designed
to
function
outside
the
cell.
 Page
3
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 4
 
 The
primary
structure
of
a
protein
contains
all
the
information
needed
to
specify
 the
normal
 secondary
and
tertiary
structure
 
 This
was
demonstrated
by
Christian
Anfinsen
in
an
important
experiment
in
the
early
1960's,
to
 answer
the
question
of
how
proteins
fold
(Lehninger
p.
141
(4th
ed
p.148)).

 
 Is
some
kind
of
a
template
needed
for
the
polypeptide
to
wrap
 around
it,
or
is
the
amino
acid
 sequence
in
the
polypeptide
sufficient
to
guide
folding
with
no
external
assistance?

 
 Starting
with
the
enzyme
 ribonuclease,
a
small
protein
of
124
amino
acids,
the
enzyme
is
first
 treated
 with
 urea,
 NH2CONH2.
 Concentrated
 urea
 solutions
 weaken
 the
 hydrophobic
 interactions,
causing
the
protein
to
unfold
or
 denature
and
lose
ability
to
act
as
an
enzyme.
In
 addition,
 ribonuclease
 contains
 four
 disulfide
 bonds
 between
 cysteine
 side
 chains,
 in
 specific
 pairs
 which
 are
 close
 together
 in
 the
 native
 state
 of
 ribonuclease.
 The
 reducing
 agent
 2‐ mercaptoethanol
HS‐CH2‐CH2‐OH
is
added
to
reduce
‐S‐S‐
bonds
to
individual
‐SH
groups.
 
 Page
4
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 5
 
 Anfinsen
 demonstrated
that
unfolded
ribonuclease
could
spontaneously
refold
if
urea
was
first
 removed
to
allow
refolding,
and
then
the
sample
was
exposed
to
air
to
allow
disulfides
to
 re‐ form.
The
refolded
ribonuclease
regains
enzyme
activity,
and
forms
the
same
pairs
of
disulfides
 as
 the
 original,
 indicating
 that
 the
 correct
 tertiary
 structure
 formed
 itself
 simply
 from
 the
 positions
of
specific
amino
acids
in
the
primary
structure
or
sequence.
 
 If
the
unfolded
ribonuclease
is
exposed
to
air
before
removal
of
urea,
 disulfides
come
together
 before
 the
 correct
 tertiary
 structure
 forms,
 but
 pair
 up
 at
 random,
 so
 the
 polypeptide
 can’t
 arrange
itself
in
the
structure
needed
to
behave
as
an
enzyme.
 
 Binding
and
recognition
of
other
molecules.
 
 Proteins
fold
because
 one
part
of
the
polypeptide
 chain
 binds
 and
 recognises
 another
 specific
 part
 of
 the
 same
 polypeptide.
 Exactly
 the
 same
 types
 of
 non‐covalent
 interactions
 that
 cause
 a
 protein
 to
 fold
 can
 also
 be
 used
 to
 allow
 a
 protein
 molecule
 bind
 any
 other
 kind
 of
 molecule.
 Strong
 binding
 interactions
 require
 a
 complementary
 match
 between
 protein
 and
 bound
 molecule,
 so
 that
 binding
 is
 highly
 specific,
 and
 serves
 as
 a
 molecular
recognition
mechanism.
 
 Nonpolar
patches
on
the
protein
surface
bind
by
hydrophobic
effect
 
 Complementary
shapes
maximize
close
contact
‐
van
der
Waals
effect
 
 Charged
or
H‐bonding
groups
line
up
with
complementary
partners,
e.g.
Donor
with
 acceptor,
+ve
with
–ve.
 
 Examples

 Proteins
which
have
 quaternary
structure
bind
other
protein
molecules
of
the
same
 type
to
 form
multiprotein
complexes.
 Enzymes
are
proteins
that
bind
and
identify
other
molecules
and
catalyze
a
specific
reaction.
 Antibodies
are
proteins
that
bind
and
recognize
foreign
molecules,
e.g.
on
invading
 bacteria
 or
viruses,
and
tag
them
for
attack
by
other
components
of
the
immune
system.
 
 Proteins
have
very
diverse
shapes,
making
them
ideal
to
form
regions
on
their
surface
which
 match
and
complement
a
particular
target
molecule.
 Page
5
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 6
 
 Each
protein
has
a
unique
shape
and
can
bind
specific
target
molecules.

 
 Binding
and
Bonding,
correct
terminology:
These
terms
may
seem
interchangeable
in
everyday
 speech,
 but
 not
 in
 biochemistry.
 If
 a
 protein
 binds
 another
 molecule,
 the
 interaction
 is
 non‐ covalent
 and
 usually
 freely
 reversible.
 Covalent
 links
 between
 two
 parts
 of
 a
 molecule
 are
 referred
to
as
bonding.
 
 
 Example
 ‐
 binding
 and
 recognition
 of
 target
 peptides
 by
 peptidases
 of
 the
 chymotrypsin
 family:
 
 peptidase
 =
 generic
 name
 for
 enzymes
 that
 catalyze
 hydrolysis
 of
 peptide
 bonds.
 Chymotrypsin
 and
 a
 number
 of
 related
 enzymes
 share
 a
 common
 core
 structure
 responsible
for
binding
the
target
peptide.
 
 A
 groove
 in
 the
 chymotrypsin
 molecule
 accommodates
 a
 target
 peptide
 chain,
 and
 forms
hydrogen
bonds
to
its
backbone
CO
and
 NH
 groups.
 This
 interaction
 is
 very
 similar
 to
 binding
an
extra
b‐sheet
strand
of
 its
own.
In
 the
figure,
the
components
of
the
enzyme
are
 in
 red,
 and
 the
 components
 of
 the
 target
 molecule
are
in
black.
 
 A
hydrophobic
pocket
in
the
chymotrypsin
fits
 a
 side
 chain
 with
 an
 aromatic
 ring,
 e.g.
 Phe,
 Tyr
 or
 Trp.
 Binding
 of
 the
 target
 peptide
 is
 much
 tighter
 if
 this
 pocket
 is
 occupied
 by
 an
 appropriate
amino
acid,
and
this
positions
the
 peptide
bond
on
the
carboxylate
side
of
the
 aromatic
 amino
 acid
 next
 to
 the
 catalytic component
of
chymotrypsin,
which
proceeds
 to
hydrolyze
that
bond.
 Page
6
of
7
 BIOC*2580
Lecture
12.
 Protein
Stability,
Binding
and
Recognition.
 7
 
 
 The
 enzyme
 Elastase
 is
 similar
 to
 chymotrypsin,
 but
 in
 elastase,
 part
 of
 the
 pocket
 is
 blocked
 off
 by
 a
 pair
 of
 bulky
 valines.
 The
 best
 fit
 for
 the
 target
peptide
occurs
if
it
places
alanine
or
glycine
 in
the
binding
 
 Page
7
of
7
 
 The
 binding
 pocket
 is
 formed
 by
 amino
acids
Gly,
Trp
and
Leu
of
the
 chymotrypsin
 molecule
 (shown
 in
 red),
 which
 have
 hydrophobic
 properties
to
match
the
target.
 
 Trypsin
 is
 similar,
 but
 the
 pocket
 is
 narrower,
 and
 the
 trypsin
 has
 a
 negatively
charged
 aspartate
in
the
 back
 of
 the
 pocket.
 The
 target
 peptide
now
 fits
best
in
the
binding
 pocket
with
an
amino
acid
having
 a
 long
 side
 chain
 with
 a
 positive
 charged
at
the
end;
hence
Lysine
or
 Arginine
are
bound
 and
recognized,
 and
 hydrolysis
 occurs
 on
 the
 next
 peptide
bond.
 
 ...
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