Conforma(onal
Sampling


Problem:

How
to
find
all
of
the
possible
conforma(ons
for
a

flexible
molecule
(protein,
nucleic
acid,

polysaccharide,
ligand,
drug)



The
selected
approach
will
depend
on
several
things

including:

1)  The
size
of
the
molecule,
and
par(cularly
the

number
of
expected
conforma(onal
states

2)  The
ability
to
define
the
states
in
obvious
internal

coordinates,
such
as
torsion
angles

 Conforma(onal
States


How
to
find
the
stable
states
(conforma(ons)
of
a
molecule?

What
defines
the
state
(or
conforma(on)
as
“stable”?





 Using
Grid
Searching
to
Find
Conforma(onal
States

If
the
states
are
related
by

simple
internal
coordinates,

such
dihedral
angles,
the
states

can
be
found
by
searching
all
of

the
dihedral
angle
space.


 N
360
I.e.
vary
the
dihedral
angle
and
 The
number
of
conforma(ons
 = ∏
i=1
θi
look
for
low
energy
structures
–

this
is
known
as
Grid
Searching

€
 Grid
Searching
and
Combinatorial
Explosions

Number
of
 Step
size
 Number
of
 Total
number
of

rotatable
 (angle
 conforma8ons
to
 conforma8ons

bonds
 increment)
 generate

1
 10
 360/10
=
36
 36
 The
principle

1
 30
 360/30
=
12
 12
 problem
with
Grid

Search
methods
is

2
 30
 =
12*12
 144
 that
the
number
of

3
 30
 =
12*12*12
 1,728
 structures
to
be

4
 30
 =
12*12*12*12
 20,736
 evaluated
increases

rapidly
–
this
is
the

5
 30
 =
12*12*12*12*12
 248,832
 “Combinatorial

6
 30
 =
125
 2,985,984
 Explosion”
problem


N
360 If
it
takes
1
second
to
compute
the

The
number
of
conforma(ons
 = ∏
i=1
θi energy
of
each
conforma(on,
how

many
days
will
it
take
to
perform
a

Grid
Search
of
6
bonds?

€
 Stochas(c
Conforma(onal
Sampling

An
alterna(ve
to
Grid
Searching,
is
to
generate
structures
by
randomly
changing
the

atomic
posi(ons
either
in
Cartesian
space,
or
in
torsion
space.

Random
methods
are
also

known
as
Stochas(c
Sampling
methods.





The
ini(al
structures
are
usually
energy
minimized
and
then
sorted
with
some
sort
of

energy
cut‐off.

I.e.
Only
low‐energy
conforma(ons
are
kept
–
but
the
choice
of
what
is

“low‐energy”
is
arbitrary.

Ocen
10
–
20
kcal/mol
above
the
minimum.

All
others
are

rejected.


2.5

The
user
decides
how
many
random
structures

2.0
 to
generate.


1.5
 

For
this
reason,
Stochas(c
Sampling
can
be

1.0

much
more
efficient
than
grid
searching,
since

0.5
 it
can
avoid
the
Combinatorial
Explosion

0.0
 problem.

0
 30
 60
 90
 120
 150
 180
 210
 240
 270
 300
 330
 360

Stochas(c
Conforma(onal
Sampling

Stochas(c
Conforma(onal
Sampling

 Stochas(c
Conforma(onal
Sampling


Both
Stochas(c
and
Systema(c

Searching
work
“OK”
for
small

molecules

 Levinthal’s
Paradox
–
Why
Nature
can’t
use
Grid

Searching
to
Fold
a
Protein

In
1969,
Cyrus
Levinthal
noted
that,
because
of
the
very
large
number
of
degrees
of
freedom
in
an

unfolded
polypep(de
chain,
the
molecule
has
an
astronomical
number
of
possible
conforma(ons

[1].



For
example,
a
polypep(de
of
100
residues

will
have
99
pep(de
bonds,
and
therefore
198
different

phi
and
psi
bond
angles.
If
each
of
these
bond
angles
can
be
in
one
of
three
stable
staggered

conforma(ons,
the
protein
may
fold
into
a
maximum
of
3198
different
conforma(ons.




If
a
protein
were
to
amain
its
correctly
folded
configura(on
by
sequen(ally
sampling
all
the
possible

conforma(ons
(i.e.
by
Grid
Searching),
it
would
require
a
(me
longer
than
the
age
of
the
universe
to

arrive
at
its
correct
na(ve
conforma(on.




This
is
true
even
if
conforma(ons
are
sampled
at
rapid
(nanosecond

or
picosecond)
rates.
The

"paradox"
is
that
most
small
proteins
fold
spontaneously
on
a
millisecond
or
even
microsecond
(me

scale.




The
fact
that
many
naturally‐occurring
proteins
fold
reliably
and
quickly
to
their
na(ve
state
despite

the
astronomical
number
of
possible
configura(ons
has
come
to
be
known
as
Levinthal's
Paradox.



Levinthal,
Cyrus
(1969).
"How
to
Fold
Graciously".
Mossbauer
Spectroscopy
in
Biological
Systems:
Proceedings
of
a
mee(ng
held
at

Allerton
House,
Mon(cello,
Illinois:
22–24.


 Conforma(onal
States


2.5


2.0


Energy
 1.5

(kcal/mol)

1.0


0.5


0.0

0
 30
 60
 90
 120
 150
 180
 210
 240
 270
 300
 330
 360


Torsion
Angle
(degrees)


What
defines
the
state
(or
conforma(on)
as
“stable”?

At
a
given
temperature
which
states
are
likely
to
be

populated?


 Conforma(onal
States
Depend
on
Temperature


2.5


2.0


Energy
 1.5

(kcal/mol)

1.0


0.5


0.0

0
 30
 60
 90
 120
 150
 180
 210
 240
 270
 300
 330
 360


Torsion
Angle
(degrees)


Average
Kine(c
Energy
=
3/2kBT

kB
=
Boltzmann’s
constant
=
0.001 987
kcal/mol/K

At
300K
how
much
kine(c
(thermal)
energy
is
available

to
a
molecule?

 Which
Conforma(onal
States
Are
Relevant?


2.5

360

330

2.0
 300

270

Energy
 240

1.5
 210

(kcal/mol)
 180

150

1.0
 120

90

60

0.5
 30

0

0.0

0
 30
 60
 90
 120
150
180
210
240
270
300
330
360

Simula(on
Time

Torsion
Angle
(degrees)


Not
all
possible
states
will
be
populated
(observed)
at
room

temperature

For
this
reason
room‐temperature
MD
is
inefficient
at
finding

conforma(onal
states



 Which
Conforma(onal
States
Are
Relevant?


20.0

18.0
 360

330

16.0
 300

14.0
 270

240

Energy
12.0
 210

180

(kcal/mol)
10.0
 150

8.0
 120

6.0
 90

60

4.0
 30

2.0
 0

0.0

0
 30
 60
 90
 120
150
180
210
240
270
300
330
360

Simula(on
Time

Torsion
Angle
(degrees)


By
raising
the
temperature
it
is
possible
to
find
other
states

This
approach
can
be
employed
in
either
MD
simula(ons
or

MC
sampling



 Increasing
Temperature
Increases
Sampling


State


Simula(on
Time


Increasing
the
temperature
will
enable
more
states
to
be
detected

during
the
simula(on
–
this
is
known
as
Simulated
Annealing



But
for
how
long
should
the
simula(on
be
run?


To
what
temperature
should
the
system
be
heated?

 Lowering
Internal
Barriers
Increases
Sampling


2.5
 2.5


2.0
 2.0


Energy
 1.5
 1.5


(kcal/mol)

1.0
 1.0


0.5
 0.5


0.0
 0.0

0
 30
 60
 90
 120
150
180
210
240
270
300
330
360
 0
 30
 60
 90
 120
150
180
210
240
270
300
330
360


Torsion
Angle
(degrees)


An
alterna(ve
to
raising
the
energy
is
to
lower
the
barriers

But
how
do
you
know
what
barriers
to
lower?

Must
be
able
to
iden(fy
simple
internal
coordinates
that
are
related
to

the
states,
such
as
torsion
angles

Conforma(onal
Sampling
with
Reduced
Barriers

Conforma(onal
Sampling
with
Reduced
Barriers

Conforma(onal
Sampling
with
Reduced
Barriers

Conforma(onal
Sampling
with
Reduced
Barriers

 Choice
of
Conforma(onal
Sampling
Method


Thus
the
problem
of
conforma(onal
sampling
is
different
for
a
small

molecule
(with
few
rotatable
bonds)
than
for
a
macromolecule,
such
as
a

protein



Small
molecule
–
can
use
Grid
or
Stochas(c
Searching
to
generate
an

ensemble
of
structures



Macromolecule
–
use
Simulated
Annealing,
or
Monte
Carlo
(MC)
Sampling,

or
long
MD
simula(ons



In
the
limit
–
that
is,
once
all
of
the
stable
states
have
been
iden(fied
and

their
popula(ons
weighted
by
their
rela(ve
energies
–
each
method
should

give
the
same
answer
–
this
is
related
to
the
“Egrodic
Hypothesis”



Ergodic
Hypothesis:
the
(me
average
property
(from
MD)
is
the
same
as
the

ensemble
average
property
(from
MC)