Quasielastic light scattering by biopolymers. Conformation of chromatin multimers

BIOCHEMICAL

Vol. 73, No. 2,1976

AND BIOPHYSICAL

Quasielastic Light Scattering Biopolymers. Conformation Chromatin Multimers*

Barbara Ramsay Shaw Department of Chemistry Duke University Durham, North Carolina

RESEARCH COMMUNICATIONS

by of

27706

and Kenneth S. Schmitz Department of Chemistry University of Missouri-Kansas Kansas City, Missouri 64110 Received

July

City

26,1976

Abstract Chicken erythrocyte chromatin was partially digested with micrococcal nuclease and separated into multimeric subunit fractions by gel permeation chromatography. The fractions were characterized by their Svedberg constant, diffusion coefficient, circular dichroism, and electrophoresis pattern of the extracted DNA. The molecular weight dependence of the sedimentation coefficient was found to be S M-554. $:'Hh: K:::w:od The molecular weight dependence of f/f, is best represented theory by either a helical superstructure or a flexible coil with attractive ThFdimer calculations of f/f, suggest that interactions between nucleosome units. theeparticles are separated by spacer regions which contribute up to -20% of the frictional properties of the molecule. Introduction Partial (5)

nuclease

and other

periodically et al.

eukaryotes organized

(6, along

( 9) independently

of the histones (10-14) core

digestion

suggests particles,

wrapped

around

"beads-on-a-string" * Supported Research

of chromatin 7) suggests the

proposed

from mammals (l-3),

that

chromatin

strand.

models

i.e., the

40-60

base pairs

a DNA-histone eight-histone structure

in part by grants Corporation.

the repeat

complex as observed

(9,

15,

by electron

from NSF (GP-25551

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

lb),

224

and two each evidence

DNA serve

to link

base pairs

thereby

are

Chemical

unit.

-140

plants

and Van Holde

DNA strand

accessible

in which

(4),

regions (8)

a short

of nuclease

complex

resistant Kornberg

in which

H2A, H2B, H3 and H4 comprise that

nuclease

yeast

giving

microscopy

and BMS 73-06819

of DNA is rise

(17-20). A02)

and

to a

BIOCHEMICAL

Vol. 73, No. 2, 1976

We report

herein

mers on molecular were

used

employed

in the

Bloomfield lattices.

a helix

friction

the

random

flight

of nucleosomes

helix,

using

of our

with

theory

was used

and,

polymer

multi-

hydrodynamic f/f,

values

of Filson

tetrahedral that

obtaining

interactions

for

results

space,

methods

(S) coefficients

to compute

suggest

compact,

attractive

(27)

the

in a free

investigations is

of chromatin

and sedimentation

on a string

in solution

coil

properties

(D) and Svedberg

The Kirkwood

polygon,

RESEARCH COMMUNICATIONS

scattering

the diffusion

beads

The results

or flexible

light

analysis.

planar

(21),

structure

Quasielastic

functional

regular

of hydrodynamic

respectively,

between

rod,

gonal

weight.

to obtain,

interaction rigid

the dependence

AND BIOPHYSICAL

the

and and hexa-

supramolecular

conformations

between

core

for

of either

particles.

Experimental -The preparation viously

(11,

was carried cooling

and isolation

12).

Digestion

of the nuclei

out at 37°C and terminated

on ice.

The nuclei

were

in 10 ml of lOmM TrisaHCl, on a Virtis

minutes

and the

homogenizer.

column,

90 x 2.5 cm, equilibrated

supernatant

fraction

Sedimentation at 30,030 scattering gravity-flow throughout temperature tial

decay

operating

rpm.

where

D is

were

facility

into

the

collection similar

clip

the solution

at 12000xg

debris

obtained

scattering interval

function, mode,

gradient

with

decay

A-5m

light Samples

by circulating

a Saicor constant

5°C.

E ultracentrifuge

(22-24).

(23).

15

centrigugatior

by quasielastic

described

computed

for

to a Bio-Rad

at 5°C and temperature

was effected

has a homodyne

at 10,OOOxg

sucrose

described

previously

at medium

0.7mM EDTA, pH 7.5 at

determined

cells

resuspended

30 seconds

on a Beckman Model

were

(Worthington)

15 minutes,

and applied

by isopycnic

previously

to that

for

pre-

1OmM in EDTA and

was pelleted

1OmM Tris'HCl,

coefficients

autocorrelation

l/T

with

coefficients

the data

in the

Nuclear

described

nuclease

0.7mM EDTA and disrupted

purified

filtered

jacket

by making

was further

on the

were

by micrococcal

was made 7% in sucrose

Diffusion

methods

erythrocytes

centrifuged

pH 7.5,

setting

The dimer

of chicken

were

control ice

water

The single

in a

exponen-

43A autocorrelator l/r

given

by (25)

= 2D(4=n/h,)2sin2(0/2)

the d iffusion

coefficient,

(1.) n is

225

the

index

of refract

ion,

X, = 488 nm,

Vol. 73, No. 2,1976

BIOCHEMICAL

t Figure

least-squares

where

analysis

the

scattering

C(t)

of a single

K = (4m/Ag)sin(B/2)

and 0 is

Function

functions

C(t)

in the

present

exponential

decay

= A exp(-2DK2t) (cf.

angle.

of Chromatin

text

Multimers. study

with

were

analyzed

by

baseline,

+ B

for

definition).

A representative

(40 --<8<70) *

curve

is

given

in

1. Representation -The molecular

weight

was computed

M = (S/D)RT where for

(m,ll,seconds)

Autocorrelation

The autocorrelation

Data

RESEARCH COMMUNICATIONS

1 Representative

Figure

AND BIOPHYSICAL

S is the

in Svedbergs

chromatin

the two sets

the

Svedberg

equation,

x 10 -13/(l-;o)

(2)

and ;p = .68.

multimers

of data

from

A plot

and native

of In S vs In M is

DNA (26).

The empirical

given

in Figure

relationships

2

for

are

S = 0.011 20

M.554

(multimers)

S

M.Z80

(native

DNA)

of the

friction

(3)

and 20

The molecular Einstein

= 0.163 weight

dependence

(4) factor

ratio

f/f,,

using

the

relationship f = kT/D

(5)

226

Vol. 73, No. 2, 1976

Figure

BIOCHEMICAL

Weight

The molecular and sedimentation pH 7.5) lysine

rich

and Stoke's

nuclease

Chromatin

for

column

digested

Hl and H5).

were

computed

fractions

chicken

Multimers

(in

DNA values

(----I

s 20 = .Oll

M'554

(----)

S = .I63 20

Mm2"

and Native

from

chromatin were

DNA

the diffusion

1OmM Tris-

erythrocyte

The native

and Inman

0.7mM EDTA, (containing

reported

by Record,

(26).

equation

solvent

viscosity

expression f i,

the

of S20 for

of the multimers

coefficients

histones

n is the

theoretical

Dependence

weight

of partial

Woodbury

where

RESEARCH COMMUNICATIONS

2 Molecular

where

AND BIOPHYSICAL

and N is

of the Kirkwood

Avogadro's

theory

number,

was compared

with

the

(27)

nR = Rs(l+t

R is

a friction

bead

average

distance

between

C ) ij i#j radius, n is the number centers

of beads

227

of beads

i and j,

per

and Rs is

chain,

is 11 defined in Eq. (6)

Vol. 73, No. 2, 1976

Results

multimers

which

is

exhibit

a more compact

closely

represented

The three-dimensional

(28).

when

compared

parameters distance

are

involved

RESEARCH COMMUNICATIONS

Rs = 52;

ratio

f/f,

model

(B = 2R).

(cf.

= 1.85

radius,

apparent

inconsistency

particles

particle

ranging

"effective"

friction and spacer

20% of the

friction

was calculated of DNA in

models computed

region,

which

properties

using

of the

dimer

are presented from

values

and Bloomfield

contiguous

spheres.

in the

region

spacer

reported region,

represents

value the

an upper

in Table

I for

of u 12'

in units

(21)

and employed

n=l2.

( > f'fo = (L&4)(1 These values

(estimated

of the

limit.

region

of adjacent

coil

bead

equivalent R' = 62; The the

(RI-R)/R'). pairs

a core an

accounts

(12)

for

core for

up to

Since

62A

the length

to the frictional

Comparison

The random

from

in length.

20% contribution

between

we calculate

of both

of 40 base

core this

regions

of radius

from

bead

estimated

= 136;,

spacer

The

We interprete

contribution the

con-

contiguous

of spacer

of 112;

unknown

(S20 = 16.2).

estimates

beads

from

fraction,

The hydrodynamically

chains

that

dimer

dimer

of B at 236;,

friction

implies

inferred

a helical

18, 29-31).

friction

weights

several

for the

support

DNA

center-to-center

reported

of 40 x 3.4;

spacer the

is Since

from

15, 17,

the value

of the

the minimum

the erythrocyte

11,

evidence

frictionless represents

(7).

chromatin

molecular

of 5.3 x lo5

the

R' of R' = 62;.

bead

of native

of R = 73; in the

to "effective"

corresponds

by semiflexible

particle

properties

radius

that

diameter

weight

than

(9,

If we fix

bead

the

(i.e.,

estimated

a value

50-55:

of 50; and a spacer

therefore,

"effective"

yields

as physical

friction

first

the molecular

from

Eq.

and fiber

pitch,

is much larger

in solution.

radius

connected

value

than

that

rod at these

employing

R and B are

the dimer

This

conformation

the macrostructure

R, R,,

Eq. 6) for for

particle

dimer,

beads,

suggests

of the polynucleosomes

structures

in describing

the parameters

where

core

model

weight

as a rigid

conformation

with

B of adjacent

formation),

for

of S20 on molecular

dependence

(a = .554)

(a = .280),

f/f,

AND BIOPHYSICAL

and Discussion

The power

Filson

BIOCHEMICAL

of f/f,

calculations

separation,

for

several

were reported

by

in the equation, +(u12/2)) were converted

228

(8) to the

"spacer

region"

model

CONFORMATION

sphere

model

from

by using

The value n = 12 is an average number computed sample and the dimer weight of 5.3 x 105.

from the contiguous calculations).

2.446 2.459 2.330

and Bloomfield

(d)

by Filson

estimated (cf. rod

reported

(cl

from

calculated

(b)

study

present

12

12 12

--1.984 1.925 1.892

2.618

the

the

average

conversion

(21)

2.28

f/f,

molecular

factor

-+ .07

weight

3.8

(c) (c) (c)

of

: 3.0

3.057 3.074 2.913

(c)

(c)

the multimer

2 1.25

B = 236;

2.397 2.480 --2.365

3.4

3.8

R = 62;;;

FOR n = 12 BEADS

B = 148;;

3.019

R = 741;

MODEL COMPARISON OF f/f,

(a)

al2

lattice

Experimental (d) (M = 3.2 x 106)

Random Walk (b) Free Tetrahedral space Cubic lattice

12 12 12 12

Helix elements/turn elements/turn elements/turn elements/turn

Contact 5 6 5 4

(a) (b) (a) (b)

12

Polygon

Regular

(a)

12

n

Rod (a)

---____

-

TABLE I.

Vol. 73, No. 2, 7 976

by the

conversion

regular

polygon

factor

feature

conformations

in either

in which

with

the

with

experiment,

friction

helix

thereby

increasing

model,

which

agreement 5 x lo5

the

all

- 5 x 106

set

of parameters,

are

of the

attractive

flexible

coil

The molecular spacer

model

Assuming core

(19)

a core

we have a total partially This

5.7 x lo4

rich

histone

spacer

as the protein

regions.

weight per

"tails" weight

core

(5.3

helix bead,

out

particles

is

in good

weight

do not

for

yield rule

radius,

in the spacer

in the helical

pair

(the

x lo5

minimum

at each end since of -4.73

105.

x

difference since

Nonetheless

2 .25 x 105)

composition

of 2.10

to the presence

content

bead

range

are an outside

These

a decrease

between

friction

the

calculations

We cannot

as well

dalton

these

consistent

between

in the molecular

of -4.7".

values.

is not

as

distance

the friction

conformations

of the dimer

attributed

for

defined

can be made to coincide

can be compensated

weight

particle

pair

touch,

repre-

helix

constitute radius,

a which

by increasing

f/f,

values

the

possibility

the

higher

than that

causing

a slight

shrinkage

is

also

consistent

with

dimer

fraction

(32).

conformation.

DNA at 660 daltons

and 20 base

and

polygon

helix,

The contact

of f/f,

since

interactions

turns

to 2 times

radius

shielding,

experimental

of rod

to adequately

center-to-center

l-112

The random walk

ratio

or regular

contact

bead helix

the

used in

rod

models

the

helical

of inclination

in more hydrodynamic

the corresponding

that

to the solvent.

however,

of inclination.

or spacer

clear

between

The parameters

unique

there

also

values

to the

of the

The contiguous

experimental

limit

inability

bead

a more realistic

of 300A and an angle

angle

ths

in adjacent

exposure

RESEARCH COMMUNICATIONS

I).

by increasing

radius

results

is

to a value

employs

with

It

results.

turns

Table

I is

beads

however,

as a --lower

contiguous

data.

experimental

adjacent

(cf.

the

AND BIOPHYSICAL

obtained

of Table

the experimental

a helix

1.25,

conformations

A salient

sent

BIOCHEMICAL

and a minimum

80 base pairs

40 base pairs

for

these

are not

The remaining of lysine-rich

may be regarded

up to an additional these

of the

calculations

230

the spacer

limit-digest

-5.7 histones

as an upper 20 base

pairs

support

the

x lo4

of nonregion

samples daltons

can be

in the dimer limit

for

particle.

the

lysine-

DNA may be in the concept

that

(ll)),

lysine-

the

Vol. 73, No. 2, 1976

rich

histones

BIOCHEMICAL

are associated

with

AND BIOPHYSICAL

dimer

particles

RESEARCH COMMUNICATIONS

as postulated

(11,

32).

coefficient

on the

Conclusions 1) weight

The power

dependence

(a = . 554) suggests

of the sedimentation that

the supramolecular

structure

molecular

of the. nucleosomes

is

compact. 2) f/f,

Model

calculations

as a function

of molecular

polygon,

random

possible

conformations

3)

walk,

either

interactions 4)

Analysis regions

nucleosomes

Dr.

is

friction

interpreting

lysine-rich

(NSF GP-25551,

We are

indebted

(K.S.S.))

to Ms. Georgia

regular

(since

of

planar

f/f,+l)

as

that,

in low ionic polymer

by the chromatin f/f,

for

with

attractive

multimers.

the dimer

hydrodynamic

strength

suggests

properties

that

of poly-

histones.

K. E. Van Holde

Schurr

ratio

dependence

in our samples.

-or a flexible-coil obtained

the

rod,

structures

suggests

factor

in

to analyze

the rigid

spherical

herein

particles

are important

We thank

eliminates

conformation

core

theory

by polynucleosomes

presented

of the

containing

weight

obtained

a helical

between

the Kirkwood

and closest-packed

The calculations

solutions,

spacer

using

(NSF BMS 73-06819 for

Reidel

financial

support

and Ms. Leslie

A02,

(B.R.S.))

and Dr.

and use of their Lewis

for

technical

J. M.

facilities. assistance.

--References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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232