Vol

September

doi

ARTICLES

Evolutionary

information

for

specifying

a

protein

fold

Michael

Steve

W.

William

P.

Heather

Kevin

H.

Rama

Classical

studies

show

that

for

many

proteins

the

information

required

for

specifying

the

tertiary

structure

is

contained

in

the

amino

acid

sequence

Here

we

attempt

to

define

the

sequence

rules

for

specifying

a

protein

fold

by

computationally

creating

artificial

protein

sequences

using

only

statistical

information

encoded

in

a

multiple

sequence

alignment

and

no

tertiary

structure

information

Experimental

testing

of

libraries

of

artificial

WW

domain

sequences

shows

that

a

simple

statistical

energy

function

capturing

coevolution

between

amino

acid

residues

is

necessary

and

sufficient

to

specify

sequences

that

fold

into

native

structures

The

artificial

proteins

show

thermodynamic

stabilities

similar

to

natural

WW

domains

and

structure

determination

of

one

artificial

protein

shows

excellent

agreement

with

the

WW

fold

at

atomic

resolution

The

relative

simplicity

of

the

information

used

for

creating

sequences

suggests

a

marked

reduction

to

the

potential

complexity

of

the

protein-folding

problem

A

fundamental

tenet

of

biochemistry

is

that

the

amino

acid

sequence

of

a

protein

specifies

its

atomic

structure

and

biochemical

But

exactly

what

information

in

the

sequence

of

a

protein

is

necessary

and

sufficient

for

producing

the

fold

and

its

biological

activity

Despite

considerable

progress

in

understanding

the

mechanisms

of

protein

the

answer

to

this

fundamental

question

remains

unknown

The

main

problem

is

the

vast

potential

complexity

of

cooperative

interactions

between

amino

acids

processes

by

which

the

free

energy

contribution

of

one

residue

depends

on

those

of

other

These

amino

acid

couplings

could

be

pairwise

and

local

in

the

three-dimensional

structure

but

could

also

involve

more

complex

cooperativities

in

which

collections

of

residues

interact

through

three-way

or

higher-order

Given

that

protein

structures

are

typically

compact

and

well

proteins

could

be

dense

and

complex

networks

of

inter-atomic

interactions

requiring

specification

of

a

great

number

of

mutual

constraints

between

amino

acid

positions

to

define

the

fold

An

approach

to

defining

the

architecture

of

amino

acid

interactions

in

proteins

is

suggested

by

an

evolution-based

method

known

as

the

statistical

coupling

analysis

-LRB-

SCA

-RRB-

This

method

postulates

that

regardless

of

spatial

location

or

underlying

mechanism

the

conserved

functional

coupling

of

sites

in

a

protein

should

drive

their

mutual

Given

a

sufficiently

large

and

diverse

multiple

sequence

alignment

-LRB-

MSA

-RRB-

of

a

protein

family

the

mutual

dependencies

should

be

evident

in

the

conserved

statistical

correlations

between

amino

acid

distributions

at

sites

Application

of

the

SCA

in

several

different

protein

families

reveals

two

general

conclusions

-LRB-

-RRB-

the

global

pattern

of

coevolutionary

interactions

is

sparse

so

that

a

small

set

of

positions

mutually

coevolves

among

a

majority

that

are

largely

decoupled

and

-LRB-

-RRB-

the

strongly

coevolving

residues

are

spatially

organized

into

physically

connected

networks

linking

distant

functional

sites

in

the

structure

through

packing

interactions

Studies

involving

directed

mutagenesis

structure

NMR

dynamics

computational

and

literature

implicate

these

networks

of

coevolving

residues

in

contributing

to

core

aspects

of

protein

function

More

surprising

however

is

the

finding

of

sparseness

The

SCA

implies

an

unexpected

degree

of

simplicity

in

amino

acid

interactions

with

far

fewer

important

constraints

between

residue

pairs

than

would

be

expected

from

inspection

of

the

atomic

structure

Indeed

as

has

been

pointed

the

evolution-based

mapping

of

amino

acid

interactions

does

not

look

like

the

contact

graph

of

the

protein

structure

many

direct

packing

interactions

show

coevolution

scores

close

to

zero

and

some

distant

sites

linked

through

networks

of

coevolving

residues

are

predicted

to

be

coupled

Thus

the

SCA

mapping

provides

a

picture

of

proteins

as

sparsely

coupled

architectures

with

redundant

strong

constraints

linking

a

few

sites

and

a

great

deal

of

near-independent

variation

at

most

sites

To

test

the

overall

hypothesis

we

reasoned

that

if

-LRB-

and

only

if

-RRB-

the

information

contained

in

the

SCA

is

a

good

estimate

of

the

total

sequence

information

for

specifying

a

protein

it

should

be

possible

to

computationally

build

artificial

members

of

the

protein

family

using

no

information

except

the

SCA-based

parameters

of

sequence

conservation

and

coupling

In

principle

these

artificial

sequences

should

fold

into

a

structure

representative

of

the

family

and

should

function

in

a

manner

indistinguishable

from

their

natural

counterparts

In

this

and

the

accompanying

we

test

this

hypothesis

in

a

computationally

and

experimentally

facile

model

system

the

WW

domain

SCA-based

protein

design

We

began

by

carrying

out

the

SCA

for

an

alignment

of

members

of

the

WW

domain

family

WW

domains

are

small

independently

folding

protein

interaction

modules

that

adopt

a

curved

threestranded

b-sheet

configuration

and

bind

to

proline-containing

target

-LRB-

Fig.

-RRB-

In

the

SCA

conservation

at

each

position

in

the

MSA

is

given

in

an

energy-like

statistical

parameter

that

measures

the

deviation

of

the

observed

distribution

of

amino

acids

from

their

mean

values

found

in

all

proteins

-LRB-

Fig.

-RRB-

The

evolutionary

Howard

Hughes

Medical

Institute

and

Departments

of

and

University

of

Texas

Southwestern

Medical

Center

Dallas

Texas

USA

Present

address

The

Rockefeller

University

York

Avenue

New

York

New

York

USA

These

authors

contributed

equally

to

this

work

Nature

Publishing

Group

NATURE

Vol

September

ARTICLES

correlations

between

sequence

positions

are

extracted

by

carrying

out

a

perturbation

analysis

on

the

MSA

the

conservation

of

amino

acids

at

one

site

is

perturbed

-LRB-

usually

by

restricting

the

site

to

one

amino

acid

-RRB-

and

the

impact

of

this

perturbation

on

the

conservation

of

amino

acids

at

each

of

the

other

sites

is

measured

in

a

distinct

energylike

statistical

parameter

Supplementary

Fig.

shows

an

example

of

this

calculation

for

one

site

in

the

WW

MSA

Figure

shows

a

matrix

representation

of

statistical

coupling

values

for

five

different

site-specific

perturbations

in

the

WW

MSA

that

demonstrates

the

core

results

of

the

SCA

Perturbation

experiments

at

all

these

sites

simply

expose

the

same

redundant

pattern

of

coevolution

between

a

small

set

of

moderately

conserved

positions

Not

all

moderately

conserved

positions

are

coupled

several

other

sites

showing

similar

conservation

scores

-LRB-

for

example

positions

or

Fig.

-RRB-

show

little

coevolution

with

the

sites

chosen

for

perturbation

The

simplicity

of

these

findings

suggests

the

possibility

that

just

the

frequency

distribution

of

amino

acids

at

sites

-LRB-

conservation

-RRB-

plus

the

few

rules

of

coupling

in

the

SCA

matrix

may

amount

to

the

total

constraints

on

WW

sequences

To

test

this

idea

we

developed

two

computational

algorithms

for

designing

novel

protein

sequences

using

only

SCA

information

The

first

algorithm

tests

the

necessity

of

the

information

in

the

SCA

matrix

by

building

artificial

sequences

that

preserve

the

amino

acid

composition

at

sites

but

eliminate

all

statistical

couplings

between

sites

Thus

this

algorithm

presumes

that

conservation

can

be

adequately

described

as

an

intrinsic

property

of

each

site

To

implement

this

algorithm

we

simply

select

amino

acids

at

each

site

independently

based

on

the

observed

frequency

distribution

in

the

natural

alignment

Accordingly

the

SCA

matrix

for

an

alignment

of

such

sequences

-LRB-

termed

the

site-independent

conservation

-LRB-

IC

-RRB-

model

-RRB-

shows

no

statistical

coupling

between

positions

-LRB-

Fig.

-RRB-

The

second

algorithm

tests

the

sufficiency

of

the

SCA

matrix

by

building

artificial

sequences

that

preserve

both

the

conservation

pattern

and

the

pattern

of

statistical

couplings

These

sequences

are

built

using

a

Monte-Carlo-based

simulated

annealing

protocol

in

which

amino

acids

are

completely

shuffled

within

every

column

of

the

MSA

while

minimizing

the

difference

between

all

coupling

values

in

the

SCA

matrix

for

the

artificial

alignment

and

for

the

natural

alignment

At

convergence

this

algorithm

produces

alignments

of

novel

sequences

-LRB-

substitutions

per

sequence

-RRB-

that

display

the

same

conservation

pattern

and

also

closely

reproduce

the

pattern

of

statistical

couplings

observed

in

the

natural

alignment

-LRB-

termed

coupled

conservation

-LRB-

CC

-RRB-

model

compare

Fig.

and

e

-RRB-

Construction

of

designed

sequences

To

evaluate

the

designed

sequences

for

folding

we

constructed

libraries

of

synthetic

genes

for

expression

of

the

artificial

proteins

in

Escherichia

coli

Four

libraries

were

built

using

a

DNA-oligonucleotide-based

gene

synthesis

protocol

-LRB-

Supplementary

Fig.

-RRB-

-LRB-

-RRB-

natural

WW

sequences

-LRB-

N

-RRB-

drawn

randomly

from

the

MSA

-LRB-

-RRB-

IC

sequences

built

on

the

premise

that

conservation

is

strictly

an

intrinsic

property

of

each

site

-LRB-

-RRB-

CC

sequences

built

on

the

premise

that

conservation

is

a

distributed

property

parsed

among

sites

in

the

manner

described

by

the

SCA

matrix

and

-LRB-

-RRB-

random

sequences

-LRB-

R

-RRB-

in

which

amino

acids

at

all

sites

were

randomly

drawn

from

their

mean

frequencies

in

the

WW

MSA

The

natural

sequences

were

built

as

positive

controls

because

we

do

not

know

how

many

of

these

will

fold

when

expressed

as

recombinant

proteins

in

bacteria

The

random

sequences

are

negative

controls

they

contain

no

sitespecific

information

and

are

not

expected

to

fold

Table

shows

the

comparative

statistical

properties

of

the

designed

sequences

Natural

IC

and

CC

WW

sequences

show

a

mean

amino

acid

identity

to

all

natural

WW

domains

of

an

expected

result

because

all

of

these

sequences

contain

the

same

pattern

of

conservation

at

sites

Another

expected

finding

was

that

random

sequences

show

much

weaker

identities

to

natural

WW

domains

-LRB-

-RRB-

However

the

IC

and

CC

sequences

also

show

similar

top-hit

identities

the

per

cent

identity

to

their

closest

counterpart

in

the

natural

world

-LRB-

-LRB-

IC

-RRB-

and

-LRB-

CC

-RRB-

P

Kolmogorov

Smirnov

test

mean

s.d.

is

shown

-RRB-

This

finding

shows

that

despite

additional

constraints

in

their

design

CC

sequences

are

about

as

diverged

as

IC

sequences

by

this

measure

the

number

of

extra

constraints

arising

from

the

SCA

matrix

is

small

Experimental

analysis

of

designed

sequences

Figure

shows

a

flowchart

of

experiments

for

each

WW

protein

Proteins

were

expressed

as

fusions

purified

using

Ni

NTA

affinity

chromatography

and

subjected

to

SDS

polyacrylamide

gel

electrophoresis

-LRB-

PAGE

-RRB-

analysis

to

evaluate

expression

and

solubility

All

libraries

contained

sequences

that

expressed

poorly

despite

multiple

attempts

or

produced

insoluble

aggregates

-LRB-

Fig.

first

and

second

columns

-RRB-

these

were

scored

as

not

folded

In

Figure

SCA-based

protein

design

a

Structure

of

a

representative

WW

domain

-LRB-

Protein

Data

Bank

-RRB-

in

complex

with

a

target

peptide

-LRB-

in

stick

representation

-RRB-

The

two

canonical

tryptophans

are

shown

as

space-filling

side

chains

The

figure

was

prepared

using

b

SCA

conservation

scores

for

each

position

in

the

WW

alignment

in

arbitrary

units

of

statistical

Position

numbers

-LRB-

x

axis

-RRB-

and

the

secondary

structure

diagram

at

the

top

coincide

with

matrix

columns

in

c

e.

c

A

matrix

representation

of

statistical

coupling

values

from

perturbation

analysis

of

five

positions

-LRB-

rows

-RRB-

in

the

WW

domain

MSA

d

The

matrix

for

an

alignment

of

IC

sequences

built

by

randomly

selecting

amino

acids

at

each

site

from

the

observed

frequency

distributions

in

the

natural

alignment

e

The

matrix

for

an

alignment

of

CC

sequences

derived

from

a

design

algorithm

where

both

the

conservation

pattern

and

the

pattern

of

statistical

couplings

in

the

natural

alignment

are

preserved

Scale

bar

shows

the

SCA

coevolution

score

ranging

from

-LRB-

blue

-RRB-

to

-LRB-

red

-RRB-

Nature

Publishing

Group

ARTICLES

NATURE

Vol

September

Table

Statistical

properties

of

natural

and

artificial

WW

domains

WW

library

Identity

to

natural

WW

domains

in

MSA

-LRB-

mean

s.d.

-RRB-

Identity

to

closest

natural

WW

domain

in

MSA

-LRB-

mean

s.d.

-RRB-

Number

of

sequences

Natural

-LRB-

folded

-RRB-

IC

CC

-LRB-

folded

-RRB-

Random

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Drawn

randomly

from

the

natural

WW

MSA

Created

by

randomly

selecting

amino

acids

at

each

site

from

the

observed

frequency

distribution

at

that

site

in

the

natural

WW

MSA

Created

by

Monte-Carlo

simulation

Created

by

randomly

selecting

amino

acids

at

each

site

from

their

overall

mean

frequencies

in

the

WW

MSA

addition

all

libraries

contained

some

fraction

of

well-expressed

and

soluble

proteins

-LRB-

Fig.

third

column

-RRB-

These

were

further

evaluated

using

thermal

denaturation

experiments

and

for

evidence

of

a

native

state

as

described

below

Expression

data

for

all

synthetic

proteins

are

provided

in

Supplementary

Fig.

A

hallmark

of

natively

folded

small

proteins

is

cooperative

and

reversible

transition

between

folded

and

unfolded

states

In

the

WW

domain

this

folding

equilibrium

and

the

consistency

of

the

two-state

approximation

have

been

well

Here

we

followed

the

folding

reaction

by

monitoring

the

fluorescence

of

a

buried

tryptophan

-LRB-

Trp

-RRB-

which

becomes

quenched

due

to

solvent

exposure

upon

thermal

denaturation

and

therefore

reports

the

fraction

of

protein

folded

as

a

function

of

temperature

-LRB-

for

example

Fig.

c

-RRB-

We

tested

all

well-expressed

and

soluble

proteins

from

the

four

sequence

libraries

for

cooperative

and

reversible

thermal

transitions

-LRB-

Supplementary

Fig.

-RRB-

a

representative

sampling

of

these

data

is

shown

in

Fig.

Natural

WW

domains

show

a

range

of

thermal

denaturation

profiles

-LRB-

Fig.

-RRB-

Some

such

as

are

clearly

well

folded

showing

a

cooperative

denaturation

with

thermodynamic

parameters

typical

for

WW

domains

-LRB-

va

n't

Hoff

enthalpy

VH

-LRB-

DH

u

-RRB-

kcal

T

m

where

T

m

is

the

melting

temperature

-RRB-

Others

such

as

cooperatively

denature

but

are

VH

less

stable

-LRB-

DH

u

kcal

T

m

-RRB-

Still

others

such

as

despite

good

solubility

show

no

convincing

evidence

of

a

native

state

given

the

experimental

conditions

of

the

assay

To

provide

independent

support

for

categorization

of

these

denaturation

profiles

as

folded

or

not

folded

we

collected

spectra

for

a

representative

sampling

of

natural

WW

proteins

Characteristic

features

of

folded

WW

proteins

include

good

chemical

shift

dispersion

of

peaks

corresponding

to

backbone

amide

protons

-LRB-

d

parts

per

million

-LRB-

p.p.m.

-RRB-

-RRB-

often

accompanied

by

distinct

chemical

shifts

of

the

two

indole

nitrogen

protons

down-field

of

p.p.m.

-LRB-

the

two

canonical

Trp

residues

are

in

distinct

chemical

environments

one

in

the

core

and

one

solvent

exposed

see

Fig.

-RRB-

and

up-field

chemical

shifts

-LRB-

d

p.p.m.

-RRB-

corresponding

to

sidechain

methyl

Spectra

for

-LRB-

Fig.

-RRB-

and

-LRB-

Fig.

-RRB-

confirm

that

these

are

indeed

natively

folded

As

suggested

by

thermal

melts

the

NMR

spectrum

for

-LRB-

Fig.

-RRB-

shows

of

these

hallmarks

consistent

with

an

unfolded

protein

On

the

basis

of

the

analysis

of

natural

WW

sequences

we

determined

rigorous

criteria

for

folding

artificial

sequences

were

declared

as

folded

only

if

they

showed

cooperative

and

reversible

thermal

denaturation

and

H-NMR

spectra

consistent

with

a

native

state

-LRB-

Supplementary

Figs

and

-RRB-

Figure

g

i

shows

similar

data

for

a

representative

set

of

CC

sequences

Some

such

as

-LRB-

Fig.

-RRB-

are

like

soluble

but

not

folded

by

either

criterion

Others

such

as

-LRB-

Fig.

-RRB-

or

-LRB-

Fig.

-RRB-

show

thermal

denaturation

profiles

that

fall

into

the

same

range

as

natural

WW

domains

-LRB-

DH

VH

kcal

u

T

m

DH

VH

kcal

T

m

-RRB-

u

and

show

strong

evidence

in

spectra

of

being

natively

folded

In

contrast

no

IC

sequences

showed

evidence

of

native

folding

Figure

shows

thermal

denaturation

profiles

for

every

soluble

IC

sequence

-LRB-

n

grey

-RRB-

and

Fig.

l

shows

corresponding

H-NMR

spectra

for

the

three

IC

sequences

that

constitute

the

best

case

scenarios

by

thermal

melts

The

data

demonstrate

that

some

CC

sequences

are

natively

folded

with

thermodynamic

properties

similar

to

that

of

natural

WWdomains

and

that

no

IC

sequences

are

natively

folded

Figure

summarizes

the

experimental

analysis

of

all

WW

Figure

Experimental

evaluation

of

artificial

proteins

-LRB-

part

-RRB-

a

A

flowchart

of

experiments

for

evaluating

WW

sequences

Each

sequence

was

expressed

as

a

fusion

in

E.

coli

tested

for

solubility

and

if

soluble

subjected

to

thermal

denaturation

and

in

most

cases

b

SDS

PAGE

analysis

of

WW

sequences

drawn

from

four

different

sequence

libraries

N

natural

CC

coupled

conservation

model

IC

site-independent

conservation

model

R

random

Lanes

represent

pellet

or

insoluble

fraction

-LRB-

P

-RRB-

soluble

fraction

-LRB-

S

-RRB-

flow-through

after

Ni

NTA

affinity

chromatography

-LRB-

F

-RRB-

and

eluted

proteins

-LRB-

E

-RRB-

Nature

Publishing

Group

NATURE

Vol

September

ARTICLES

sequences

tested

Sixty-seven

per

cent

of

natural

WW

sequences

were

folded

by

the

criteria

described

above

a

number

that

simply

reflects

the

efficiency

of

producing

randomly

chosen

WW

domains

in

our

expression

system

As

expected

no

random

sequences

were

folded

although

nearly

half

of

these

were

well

expressed

and

soluble

No

IC

sequences

were

natively

folded

although

a

substantial

fraction

-LRB-

-RRB-

was

soluble

The

finding

that

these

sequences

showed

a

greater

probability

of

being

soluble

in

comparison

to

random

sequences

suggests

that

conservation

taken

at

sites

alone

may

provide

enough

information

about

the

folding

process

to

permit

hydrophobic

collapse

to

a

molten

globule-like

state

However

the

siteindependent

conservation

model

is

insufficient

to

produce

the

native

state

In

contrast

over

one-quarter

of

the

CC

sequences

were

natively

folded

Figure

compares

the

unfolding

enthalpy

and

melting

temperatures

derived

from

thermal

denaturation

experiments

for

all

folded

natural

WW

domains

and

CC

sequences

showing

that

the

CC

sequences

fall

into

the

same

range

of

thermodynamic

parameters

as

their

natural

counterparts

-LRB-

P

multivariate

analysis

of

variance

-LRB-

ANOVA

-RRB-

-RRB-

Just

like

natural

WW

domains

the

CC

domains

are

marginally

stable

folds

in

which

a

fine

balance

of

opposing

forces

probably

accounts

for

the

distinction

between

folded

and

non-folded

states

Folding

to

a

native

state

involves

efficient

packing

of

hydrophobic

atoms

within

the

interior

of

proteins

a

factor

that

leads

to

higher

average

sequence

conservation

in

the

core

of

Although

CC

sequences

show

similar

divergence

from

natural

WW

domains

as

IC

sequences

overall

-LRB-

Table

-RRB-

we

wondered

whether

CC

sequences

might

natively

fold

because

they

are

more

similar

to

WW

domains

within

the

core

To

examine

this

we

calculated

sequence

identities

within

core

residues

-LRB-

-RRB-

between

CC

or

IC

sequences

and

natural

WW

domains

CC

sequences

show

mean

and

top-hit

identities

to

natural

sequences

for

these

positions

-LRB-

mean

s.d.

-RRB-

values

that

reflect

the

near

invariance

of

some

core

positions

in

the

MSA

However

IC

sequences

are

no

different

from

CC

sequences

showing

mean

and

top-hit

identities

to

natural

sequences

for

core

positions

-LRB-

mean

s.d.

-RRB-

Also

the

folded

subset

of

CC

sequences

shows

the

same

core

sequence

identity

-LRB-

mean

and

tophit

mean

s.d.

-RRB-

Thus

native

folding

in

CC

sequences

is

not

explained

by

a

more

natural-like

composition

of

core

residues

Taken

together

the

data

argue

that

in

addition

to

the

amino

acid

distributions

at

sites

the

statistical

coupling

information

is

necessary

and

sufficient

to

specify

native

folding

Because

no

information

about

the

WW

domain

except

the

coupling

values

in

the

SCA

matrix

was

used

to

constrain

the

CC

sequences

beyond

the

IC

sequences

we

conclude

that

the

specific

topology

of

mutual

constraints

between

sites

predicted

by

the

SCA

is

one

solution

for

achieving

the

folded

state

of

this

protein

family

Atomic

structure

of

an

artificial

WW

domain

Do

the

artificial

proteins

adopt

the

canonical

WW

fold

To

examine

this

we

pursued

in-depth

structural

analysis

of

one

of

the

CC

proteins

shows

mean

identity

and

top-hit

identity

to

natural

WW

sequences

values

that

are

near

the

average

Figure

Experimental

evaluation

of

artificial

proteins

-LRB-

part

-RRB-

Thermal

denaturation

studies

and

corresponding

spectra

for

a

sampling

of

WW

sequences

drawn

from

the

natural

-LRB-

a

d

f

-RRB-

CC

-LRB-

b

g

i

-RRB-

or

IC

model

-LRB-

c

j

l

-RRB-

alignments

For

natural

and

CC

sequences

three

sequences

are

shown

that

were

highly

stable

-LRB-

d

g

-RRB-

moderately

stable

-LRB-

e

h

-RRB-

and

unfolded

-LRB-

f

i

-RRB-

For

IC

sequences

melts

of

every

soluble

sequence

-LRB-

N

-RRB-

are

overlaid

in

grey

spectra

of

three

sequences

-LRB-

in

black

-RRB-

with

the

best

potential

for

some

native

structure

are

shown

in

j

l.

Whereas

natural

and

CC

sequences

include

some

that

are

folded

no

IC

sequences

are

folded

Fluorescence

is

reported

in

counts

Nature

Publishing

Group

ARTICLES

NATURE

Vol

September

for

both

CC

and

IC

sequences

-LRB-

Table

-RRB-

As

described

this

protein

is

well

folded

by

several

independent

biophysical

assays

-LRB-

Fig.

g

-RRB-

We

solved

the

three-dimensional

structure

of

by

solution

NMR

methods

using

distance

and

dihedral

angle

restraints

-LRB-

Fig.

b

see

also

Supplementary

Table

-RRB-

was

refined

to

reasonably

high

precision

clearly

confirming

the

curved

three-stranded

antiparallel

b-sheet

structure

characteristic

of

all

WW

domains

However

the

structural

similarity

of

to

other

WWdomains

seems

to

go

well

beyond

just

the

fold

level

Tertiary

structure

motifs

common

to

WW

domains

are

found

in

including

a

centrally

located

tryptophan

-LRB-

-RRB-

that

sits

upon

a

platform

of

two

proline

side

chains

-LRB-

Fig.

-RRB-

In

addition

several

sites

in

display

unusual

proton

chemical

shifts

based

on

comparison

with

the

BioMagResBank

database

of

protein

NMR

data

-LRB-

for

example

d

p.p.m.

for

Fig.

see

Supplementary

Table

for

further

examples

-RRB-

Such

unusual

shifts

arise

from

unique

tertiary

packing

that

places

protons

in

close

proximity

to

aromatic

side

chains

Comparison

of

the

chemical

shifts

of

to

those

of

two

natural

WW

domains

-LRB-

ref

-RRB-

and

-LRB-

ref

-RRB-

showed

that

all

three

proteins

display

these

same

unusual

shifts

at

analogous

positions

in

each

sequence

Thus

adopts

a

stable

WW-like

three

dimensional

structure

To

examine

how

well

the

SCA-based

design

recapitulates

the

native

structure

of

the

WW

domain

we

overlaid

the

structure

of

with

those

of

several

different

natural

WW

domains

by

minimizing

the

root

mean

squared

deviations

-LRB-

r.m.s.d.

-RRB-

of

backbone

Ca

atoms

for

all

structures

-LRB-

Fig.

-RRB-

All

seven

structures

clearly

adopt

very

similar

backbone

folds

and

no

obvious

feature

seems

to

distinguish

from

the

other

proteins

To

quantify

this

result

we

compared

the

average

pairwise

r.m.s.d.

values

for

backbone

atoms

of

all

the

natural

WW

domains

-LRB-

A

-RRB-

with

the

r.m.s.d.

values

of

from

all

the

natural

domains

-LRB-

A

-RRB-

The

difference

between

these

distributions

is

not

significant

-LRB-

P

-RRB-

demonstrating

that

is

as

similar

at

atomic

resolution

to

natural

WW

domains

as

natural

domains

are

to

each

other

Conclusions

Classical

studies

indicate

that

protein

structure

and

function

results

from

globally

minimizing

the

free

energy

of

the

polypeptide

chain

under

physiological

In

this

work

we

have

tested

a

model

for

the

specific

pattern

of

amino

acid

interactions

that

makes

up

the

global

free

energy

minimum

using

the

WW

domain

as

a

model

system

-LRB-

Fig.

c

-RRB-

The

model

is

based

on

three

core

hypotheses

-LRB-

-RRB-

that

amino

acid

interactions

specifying

the

atomic

structure

are

conserved

throughout

members

of

a

protein

family

rather

than

being

idiosyncratic

-LRB-

-RRB-

that

conservation

is

a

distributed

rather

than

siteindependent

property

because

it

fundamentally

arises

from

the

cooperativity

of

energetic

interactions

and

-LRB-

-RRB-

that

the

parsing

of

conservation

is

well

estimated

by

the

statistical

energy

function

contained

in

the

SCA

method

The

finding

that

a

significant

fraction

of

CC

sequences

are

natively

folded

whereas

IC

sequences

are

not

provides

strong

support

for

these

hypotheses

This

result

is

particularly

informative

because

CC

sequences

are

statistically

indistinguishable

from

IC

sequences

with

regard

to

sequence

divergence

from

natural

WW

domains

We

conclude

that

it

is

the

specific

distribution

of

conservation

rather

than

the

quantity

of

conservation

that

dictates

native

folding

Figure

Summary

of

experiments

on

all

natural

and

artificial

WW

sequences

a

A

pie

chart

showing

the

outcomes

of

folding

studies

for

natural

-LRB-

n

-RRB-

CC

-LRB-

n

-RRB-

IC

-LRB-

n

-RRB-

or

random

-LRB-

n

-RRB-

WW

sequences

Red

natively

folded

blue

soluble

but

unfolded

yellow

insoluble

grey

poor

expressing

b

Melting

temperatures

-LRB-

T

m

-RRB-

and

va

n't

Hoff

enthalpies

of

unfolding

for

all

folded

WW

sequences

Open

circles

indicate

natural

sequences

and

filled

circles

indicate

the

folded

CC

sequences

The

artificial

sequences

show

thermodynamic

parameters

that

fall

into

the

same

range

as

that

of

natural

WW

domains

Figure

NMR

structure

determination

of

an

artificial

WW

domain

a

Ensemble

of

ten

lowest

energy

structures

determined

for

showing

backbone

traces

of

each

structure

and

side

chains

of

selected

residues

discussed

b

Ribbon

diagram

of

the

representative

structure

of

the

ensemble

Residues

highlighted

in

CPK

or

stick

bonds

illustrate

packing

interactions

in

the

core

-LRB-

Trp

Pro

Asn

-RRB-

and

in

the

canonical

proline-binding

pocket

-LRB-

Tyr

Trp

-RRB-

c

Structure-based

alignment

of

-LRB-

blue

-RRB-

with

six

natural

WW

domains

in

white

-LRB-

-LRB-

-RRB-

-LRB-

-RRB-

dystrophin

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

-RRB-

The

data

show

that

the

adopts

a

WW-like

tertiary

structure

Nature

Publishing

Group

NATURE

Vol

September

ARTICLES

How

is

the

sparse

architecture

of

residue

interactions

suggested

by

the

SCA

consistent

with

the

fact

that

proteins

are

well

packed

and

with

the

intuitive

sense

that

residues

closer

to

each

other

in

the

structure

should

interact

more

strongly

Indeed

mutations

made

in

spatially

local

sites

-LRB-

A

apart

-RRB-

are

more

likely

to

be

thermodynamically

a

finding

that

has

been

used

to

argue

that

the

energetic

architecture

of

proteins

could

be

dense

and

local

rather

than

sparse

and

However

mutation-based

free

energy

measurements

do

not

measure

the

energetic

value

of

wild-type

residues

they

measure

the

energetic

effect

due

to

mutation

When

close

in

space

it

becomes

difficult

to

separate

the

average

spatial

correlation

of

mutation-induced

effects

in

proteins

from

the

intrinsic

interactions

between

the

wild-type

residues

in

proteins

For

example

alanine

mutations

are

often

thought

of

as

loss-of-function

mutations

in

proteins

but

these

mutations

can

cause

local

cavities

to

form

in

structures

and

the

energetic

value

of

such

mutations

includes

a

cavity-dependent

In

such

cases

it

is

not

surprising

that

pairs

of

mutations

in

local

regions

show

energetic

coupling

In

contrast

SCA-based

protein

design

is

a

massive

perturbation

experiment

in

which

the

mutagenesis

at

every

site

is

constrained

by

its

own

frequency

distribution

in

the

MSA

and

by

coupling

to

those

few

other

sites

that

evolution

shows

can

provide

compensatory

mutations

Consequently

a

large

number

of

packing

interactions

are

decoupled

in

the

design

of

CC

sequences

and

vary

independently

consistent

with

findings

that

proteins

are

generally

tolerant

to

We

suggest

that

this

experiment

provides

a

more

informative

and

extensive

test

of

the

architecture

of

amino

acid

interactions

in

proteins

Nevertheless

it

is

also

clear

that

the

density

of

atoms

within

the

core

of

proteins

is

high

similar

to

that

in

crystals

of

free

amino

At

first

glance

this

finding

seems

to

highlight

the

importance

of

local

interactions

in

proteins

to

create

this

high

level

of

order

However

recent

studies

show

that

although

the

mean

density

of

atoms

in

the

core

of

proteins

suggests

a

solid-like

arrangement

the

distribution

of

packing

densities

is

much

more

like

that

of

a

That

is

packing

is

heterogeneous

in

protein

cores

with

some

highly

ordered

regions

mixed

with

many

regions

that

are

not

as

highly

ordered

A

number

of

studies

are

now

beginning

to

add

experimental

support

for

the

heterogeneous

and

distributed

nature

of

mechanical

interactions

in

It

will

be

important

to

test

further

the

physical

model

that

emerges

from

these

studies

and

the

SCA

proteins

are

heterogeneous

energetic

architectures

with

a

network

of

coupled

residues

existing

in

the

environment

of

many

weaker

interactions

METHODS

Statistical

coupling

analysis

Statistical

coupling

analysis

was

performed

as

previously

WW

domain

sequences

were

collected

using

-LRB-

e-scores

-RRB-

from

the

non-redundant

database

of

protein

sequences

-LRB-

release

date

October

-RRB-

and

aligned

using

The

five

statistical

perturbations

used

in

this

study

-LRB-

and

-RRB-

were

chosen

by

knowledge

of

structural

or

functional

importance

in

the

WW

Protein

design

algorithms

IC

model

sequences

were

created

by

randomly

drawing

amino

acids

for

each

site

from

the

corresponding

amino

acid

frequency

distributions

in

the

natural

WW

alignment

Random

sequences

were

created

by

randomly

selecting

residues

at

every

site

from

the

overall

frequency

distribution

in

the

MSA

CC

model

sequences

were

generated

using

a

Monte-Carlo

simulated

annealing

algorithm

described

in

the

Supplementary

Methods

section

Gene

construction

and

protein

expression

Genes

encoding

artificial

sequences

were

designed

by

back-translating

designed

protein

sequences

using

E.

coli

codon

optimization

-LRB-

Vector

NTI

Suite

Informax

Inc

-RRB-

built

on

overlapping

DNA

oligonucleotides

assembled

using

the

polymerase

chain

reaction

and

cloned

into

the

expression

vector

-LRB-

provided

by

J.

Noel

-RRB-

Constructs

were

verified

by

DNA

sequencing

Proteins

were

expressed

in

-LRB-

-RRB-

cells

grown

at

in

Terrific

broth

to

an

absorbance

at

nm

of

and

induced

with

mM

IPTG

at

overnight

For

fluorescence

experiments

ml

culture

was

lysed

in

ml

binding

buffer

-LRB-

mM

TrisHCl

pH

M

NaCl

mM

imidazole

-RRB-

by

sonication

followed

by

centrifugation

and

incubation

of

the

cleared

lysate

with

ml

bed

volume

Ni

NTA

-LRB-

Qiagen

-RRB-

for

min

at

After

washing

-LRB-

three

times

with

ml

binding

buffer

-RRB-

WW

proteins

were

eluted

in

elution

buffer

-LRB-

mM

TrisHCl

pH

M

NaCl

mM

imidazole

-RRB-

Cultures

were

scaled

up

for

NMR

experiments

using

l

of

Terrific

broth

and

ml

of

affinity

resin

Thermal

denaturation

assay

Purified

proteins

were

diluted

into

ml

elution

buffer

for

a

final

WW

domain

concentration

of

mM

Protein

fluorescence

was

monitored

at

nm

-LRB-

excitation

at

nm

-RRB-

using

a

spectrofluorometer

-LRB-

Photon

Technologies

Inc.

-RRB-

outfitted

with

a

Peltier

temperature

controller

Data

were

acquired

while

changing

the

temperature

from

to

and

back

to

at

a

rate

of

with

min

equilibration

periods

at

and

Melting

curves

for

mM

free

tryptophan

were

subtracted

to

account

for

the

intrinsic

temperature

dependence

of

Trp

fluorescence

Melting

temperature

-LRB-

Tm

-RRB-

and

enthalpy

of

unfolding

at

the

Tm

were

calculated

by

fitting

the

first

derivative

of

the

denaturation

curves

to

the

differential

form

of

the

va

n't

Hoff

NMR

spectroscopy

All

NMR

spectra

were

recorded

on

and

MHz

Varian

Inova

spectrometers

One-dimensional

spectra

of

various

WW

domains

were

obtained

on

samples

containing

mM

to

mM

protein

in

mM

NaCl

mM

sodium

phosphate

buffer

-LRB-

pH

-RRB-

and

All

spectra

were

recorded

at

using

a

Watergate

for

water

suppression

For

the

chemical

shift

assignments

and

solution

structure

determination

a

combination

of

two-dimensional

double-quantum

filtered

correlation

spectroscopy

-LRB-

DQF-COSY

-RRB-

total

correlated

spectroscopy

-LRB-

TOCSY

t

mix

ms

-RRB-

and

nuclear

Overhauser

effect

spectroscopy

-LRB-

NOESY

t

mix

ms

-RRB-

were

recorded

on

mM

unlabelled

protein

samples

in

the

above

buffer

at

and

along

with

two-dimensional

TOCSY

-LRB-

t

mix

ms

-RRB-

and

NOESY

-LRB-

t

mix

ms

-RRB-

spectra

recorded

at

on

a

mM

protein

sample

made

in

the

same

buffer

with

solvent

Additional

spectra

were

recorded

on

an

mM

sample

of

uniformly

in

O

O

including

three-dimensional

TOCSY

-LRB-

t

mix

ms

-RRB-

three-dimensional

NOESY

-LRB-

t

mix

ms

-RRB-

threedimensional

HNHA

and

two-dimensional

heteronuclear

single

quantum

correlation

-LRB-

HSQC

-RRB-

experiments

All

spectra

were

processed

using

the

NMRPipe

and

analysed

using

The

sequence

is

NH

MPLPPGWERRTDVEGKVYYFNVRTLTTTWERPTIILE-COO2

Structure

determination

Distance

restraints

were

obtained

by

analysing

the

three-dimensional

NOESY

and

two-dimensional

homonuclear

NOESY

spectra

recorded

in

-LRB-

both

t

mix

ms

-RRB-

using

the

CNS

with

the

ARIA

Additional

restraints

on

the

backbone

dihedral

angles

f

and

w

were

derived

from

a

TALOS-based

analysis

of

backbone

chemical

and

supplemented

by

-LRB-

HN-Ha

-RRB-

coupling

constants

measured

in

a

three-dimensional

HNHA

spectrum

Hydrogen

bond

restraints

for

a

total

of

six

backbone

amides

were

generated

for

sites

showing

b-sheet

structure

by

dihedral

angle

analysis

and

typical

inter-strand

nuclear

Overhauser

effects

-LRB-

NOEs

-RRB-

One

hundred

structures

were

generated

in

the

last

ARIA

iteration

out

of

which

the

ten

with

the

lowest

energies

were

selected

for

a

final

refinement

stage

in

water

The

resulting

ensemble

of

ten

structures

was

used

for

all

analyses

as

implemented

in

the

programs

and

Ramachandran

statistics

for

are

in

most

favoured

regions

in

additionally

allowed

regions

in

generously

allowed

regions

and

in

disfavoured

regions

Received

January

accepted

June

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C.

B.

Principles

that

govern

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folding

of

protein

chains

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V.

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online

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paper

at

Acknowledgements

We

thank

A.

G.

Gilman

M.

Rosen

and

members

of

the

Ranganathan

laboratory

for

advice

and

critical

review

of

the

manuscript

and

E.

Olson

for

contributing

to

this

project

R.R.

and

S.W.L.

thank

the

students

of

the

Woods

Hole

physiology

course

for

participating

in

an

initial

phase

of

this

work

This

study

was

supported

by

the

Robert

A.

Welch

foundation

-LRB-

R.R.

and

K.H.G.

-RRB-

the

Mallinckrodt

Foundation

Scholar

Award

-LRB-

R.R.

-RRB-

and

the

NIH

-LRB-

K.H.G.

-RRB-

W.P.R.

is

an

associate

and

R.R.

is

an

investigator

of

the

Howard

Hughes

Medical

Institute

Author

Information

Atomic

coordinates

for

have

been

deposited

in

the

Protein

Data

Bank

under

accession

code

Reprints

and

permissions

information

is

available

at

npg.nature.com/reprintsandpermissions

The

authors

declare

no

competing

financial

interests

Correspondence

and

requests

for

materials

should

be

addressed

to

R.R.

-LRB-

rama.ranganathan@utsouthwestern.edu

-RRB-

Nature

Publishing

Group

O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

evolution
protein structure
fold
protein folding

protein sequence
protein evolution
correlated mutations