Circular dichroism of a membrane protein of Neurospora crassa

BIOCHEMICAL

Vol. 51, No. 4, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CIRCULAR DICHROISM OF A MEMBRANE PROTEIN OF VlX?O,SPC’RA

Division

of Biochemistry,

Received

February

Universitv

of Wyoming,

;7RASSA1

Laramie,

Wvoming

82071

27,1973

SUMMARY Membrane protein of a mutant of !lruros~om cransa has been isolated in aqueous sodium phosphate buffer without emploving a chemical agent such as urea, detergent, or organic solvent. The protein moved in a single band on sodium dodecyl sulfate gel electrophoresis and as well on non-SDS gel electrophoresis. The circular dichroism snectrum of the protein exhibited the characteristic low ellipticitv but no red shift. The circular resemble unique

in general anomalies.

the position Urry

(3)

that

type

dependent

light

Several

partially

authors

deaggregation increase

poly-L-glutamic and they

that

flattening

have

tried

to correct

absorption

Experimentally,

the

corrected

by sonication

(3,11),

in organic minima

were

(4)

same extent

IThis work was supported Station, Journal Article

and

.Ti and

displaved

the

the aggregation were

resnonsible

artifacts

and Rayleigh

for

upon

in different

light

in

French

the

solution

the different

However,

or Mie CD spectra

hv treating

reversed

on the theo-

on the

or in detergent

suspension.

the

have been nress

(8)

(1,12,13,14). extent

low ellipticitv

of did

experiments.

by USPHS, NIH Grant, No. 502.

Cop.vright 0 1973 b-v Academic Press, Inc. AN rights of’ reproduction in arly form reserved.

optical

distortions

solvent

always

of the membrane

the

flattening

(5-10).

to the

also

show

one-third

(1,2).

(PGA)

suggested

but

to about

wavelength acid

and absorption

of Duysens'

shifted

polypeptides

was reduced

to longer

of membranes

ellipticity.

and by solubilization The red

at minima

a-helical

in a variety

a-helical

was shifted

scattering

basis

scattering

minima

and low

of proteins

CD of right-handed

CD upon aggregation,

red shift

retical

the

spectra

The ellioticity

of the found

membrane

the

dichroism

829

CA-12853-01;

\Jvoming

Experimental

not

Vol. 51, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Another matter which complicates the interpretation ity

of the membraneproteins

average optical

activity

of the optical

activ-

is that the CD and ORDspectra represent the

of heterogeneous proteins

in membranes(15-19).

To

understand the conformation of membraneproteins and to provide an accurate experimental ground on which the distorted present it is essential

to know:

CD can be reliably

(1) to what extent optical

corrected, artifacts

distort

CD spectra and (2) not only the averaged CD of heterogeneous proteins branes but also the CD of each protein

at

in mem-

species in membranes.

EXPERIMENTAL The slime mutant of Neurospora crassa (a kind gift was grown by constant shaking in a mediumcontaining nutrient

broth,

from V. W. Woodward)

0.5% yeast extract,

2% of Vagels, and 29% sucrose at 3' for 3 days.

0.5%

Cells were

harvested by sedimentation at 4,000 x gmax for 5 min and resuspended in a grinding mediumcontaining

0.05M Tris,

0.25M sucrose, and 4mMEDTA at pH 8.5.

They

were then mixed with the samevolume of 2mmglass beads, and ground in a jar mill

for 2 hours at 5'.

The broken cells were filtered

and sedimented at 1,000 x gmax for 5 min.

They were washed five

grinding mediumand then washed in 0.05M Tris buffer no protein was detected in the supernatant.

proteins

at pH 7.5 repeatedly until

The suspension was centrifuged

at 48,000 x gmax for 2 hours to remove rough pellets.

was subject to a second centrigugation

out at 5O. The soluble proteins were fractionated

Sephadex G-100 column (2.5 x 50 cm).

The supernatant

at 314,000 x gmax for 7 hours.

remaining in the supernatant were considered soluble.

were carried

times in the

The membranepreparation was stir-

red in O.OlM Na2HP04at pH 10.3 for overnight. initially

through cheese cloth

Membrane

All procedures on an ascending

The column was eluted with 5mMNa2HP04at

pH 10.3, 0.12 ml/min, and the eluant was monitored by absorbance at 285 nm on a Heath Model 701 spectrophotometer. viously

Gel electrophoresis

was performed as pre-

described by Dunker and Rueckert (20).

Protein Poly-L-glutamic

concentration

was determined according to Lowry et.

al.

(21).

acid was purchased from New England Nuclear (Lot #G-153 and

MW38,000). 830

BIOCHEMICAL

Vol. 51, No. 4,1973

AND BIOPHYSICAL

VOLUME

RESEARCH COMMUNICATIONS

(ml]

Figure 1. Column fractionation of the soluble membrane proteins. The first peak was eluted at the exclusion volume and contained about 5% of the input proteins. The second peak contained the rest of the input nroteins.

CD spectra mm cell

was measured

was used.

to the amino

The residue

acid

content

Bowman spectrofluorometer The wavelength

on a Jasco

was used

were

was estimated personal

to determine

and scattered

well

CD/ORD spectropolarimeter.

ellipticity

(V. W. Woodward,

of incident

and fluorescence

molar

J-20

A 0.2

as 120 according

communication),

right

angle

An Aminco-

light

beam was 290 nm where

scattering. scattered

light

separated.

RESULTS AND DISCUSSION On the column fraction

(5%) and a major

peak was eluted tran

(MW 2 x 106).

volume

protein

is

manuscript

fraction

Since

of the Sephadix soluble

was 1.65

and that coefficient and the

in preparation).

soluble

(95%) volume

the major

at 315,000

The sedimentation 2.1 mg/ml

the

at the exclusion

on centrifugation sion

chromatography,

x g G-100 its

as appears which

protein

fraction

7 hours

column,

it

of the

specific

Therefore,

is reasonable is

protein volume

831

below

showed 1.

in the after

to consider 150,000

a minor

The minor

by using

remained

the molecular

daltons.

in Figure

and was eluted

weight

soluble

proteins

was determined

for

max

molecular

partial

membrane

blue

dex-

supernatant the

exclu-

that

daltons.

at the concentration was 0.75 weight

(Ji

the

of

and Woodward,

is near

18,000

Vol. 5 1, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PGA

-

MEMBRANE

PROTEIN

-

-4B 180

220

208 Ah)

Figure 2. Polyacrylamide gel electrophoresis (A) and SDS-polyacrylamide electrophoresis (B) of the fractionated soluble protein (peak 2 in Figure The gels were electronhoresed downward. Figure

3. CD spectra of a-helical PCA and the fractionated The CD spectrum of (peak 2 in Figure 1) at pH 7.7. tein was measured in 30 min after the solution was adjusted was no apparent light scattering and absorption flattening The CD spectra was obtained at the saeed of 4-2 nm/min and of l-4.

soluble membrane the membrane proThere to pH 7.7. in the sample. at the time constant

protein

Figure

2 shows

in SDS or non-SDS protein

protein column light

1 reveals

fraction

that

right

exhibited

and indicates

one band that

the

size.

pH.

about

fraction

electrophoresis

angle

and gel

increased

in the major

gel

in particle

at different

chromatography, scattering

the protein

polyacrylamide

is homogeneous Table

that

gel 1).

light

scattering

behavior

At pH 9.5 and 10.3

electrophoresis 2.5-fold

were compared

832

of

the major

where

solubilization,

performed,

the

to that

intensity

at pH 11.3

where

of

Vol.51,No.4,

BIOCHEMICAL

1973

Table

1.

Relative

intensity

membrane

orotein.

of right

Time

angle

RESEARCH COMMUNICATIONS

scattered

light

Relative

PH was

light

(hrs)

10.3

o-24

2.5

9.5

O-24

2.5

7.7

0

2.5

7.7

0.5

2.5

7.7

24

in light

scattering

reasonably and the turbid

good time

constant

and the

pH 7.7 for

of solute

light

at least

for

at

when

from

increase

is

1-4 on the

30 min which

instrument.

increased

light

if

change

was enough speed

Iiowever,

30 fold

it

to scan a

of 4-2

nm/min

became visibly

the solution

at pH 7.7

This

figure

a-helical

CD spectra. to scatter the diameter

close

was kept

light

not

CD at around

protein

is

at

at pH 7.7 should

2.5 fold

is

is

totally weight

protein

globular.

Therefore, by light

due to an becomes for

45,000

measuring

PGA does.

at pH 7.7 In

be 35 A0 versus

Since

scattering

not

in the

in solution

will

the

does

increase

PGA employed

at pH 11.3

be distorted 833

of the solute

the a-helical

the light

scattering. not

than

weight

and assuming

molecular

protein

the protein

in Rayleigh

the

of o-helical

much more

2000 A',

constant,

at pH 11.3,

the membrane

of the membrane

ted

that

the corrected to that

that

kept

to molecular

coefficient

to that

Consequently,

assuming

at pH 7.7

is

is assumed

compared

48 A0 at pH 7.7, in measuring

it

weight,

is

proportional

and the virial If

in the molecular

expected

is directly of solute

isotropic (22).

daltons.

membrane

initial

was no further

250 nm to 190 nm at the

the concentration

scattering

protein

At pH 7.7 there

the

scattering

significantly

addition,

performed.

of scattered

scattering

change

30.

24 hours.

Intensity

light

light

1

were

CD spectrum

of

scattering

o-24

studies

of the

intensitv

11.3

sedimentation

is

after

adjusted

PH

AND BIOPHYSICAL

we are

interes-

by the membrane the

CD spectra scattering

of the as the

BIOCHEMICAL

Vol. 5 1, No. 4, 1973

CD spectra for

of aggregated

such small

222nm,

but

(degrees

those

obtained

sonicated 24 hours

is

the

the data

do not

proteins

but

the

also

cell

which that

indicate

proteins

only

optical

in a cell

of

and a typical

3.

protein

appears was 1.67

of the ellipticity

are

membranes solution

similar

sheared

by French

shifted

shift

toward

is a total

x lo4

protein

is

identical press for

longer

of the

at

of a-helical

but not

at pH 7.7 was kept

is characteristic the red

that

at pH 7.7

of the CD spectra

The data

is a partial

membrane

flattening

at 192 nm of the membrane

222 nm minimum

concluded

1 mg/ml

in Figure

one half

ellipticity

blood

low ellipticity

Also,

shown

about

of PGA.

decreased, it

that

in solution

in both

As the protein

to aggregate,

Therefore, the

red

(11,14).

are

is

the

of that

from

the ellipticity

that

which

However,

one half

absorption

at 222 nm of the membrane

cm2 decimole-I)

than

less

protein

transition

ellipticity

PGA, 3.4 x 104. less

at pH 4.5,

of n-r*

the

Furthermore,

(4,8,9).

of the membrane

PGA in water

The position

were.

at a concentration

is negligible

The CD spectra cc-helical

membranes

particles

0.2 mm pathlength

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

or

longer

than

wavelength

and

CD spectra optical

(8)

to

of membranes.

artifact

and

artifact. the

low

represent activity

ellipticity

an average

a-helical optical

of some membrane

activity

CD spectra

of

of the

protein.

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

Lenard, J. and Singer, S. J., Proc. Nat. Acad. Sci. U.S.A., 16, 1828 (1966). Choules, G. L. and Bjorklund, R. F., Biochem., 2, 4759 (1970). Ji, T. H. and Urry, D. W., Biochem. Biophys. Res. Commun.,34, 404 (1969). Duysens, L. M. N., Biochim. Biophys. Acta, 19, 1 (1956). Urry, D. W. and Ji, T. H., Arch. Biochem. Bzphys., 128, 802 (1968). Ottaway, C. A. and Wetlaufer, D. B., Arch. Biochem. zphys., 139, 257 (1970). Urry, D. W. and Krivacic, J., Proc. Nat. Acad. Sci. U.S.A., 65, 845 (1970). Glaser, M. and Singer, S. J., Biochem., 10, 1780 (1971). Gordon, D. J. and Holzwarth, G., Arch. B&hem. Biophys., 142, 481 (1971). Gordon, D. J. and Holzwarth, G., Proc. Nat. Acad. Sci. U.S.A., 68, 2365 (1971). Schneider, A. S., Schneider, M.J.T. and Rosenheck, K., Proc. Nat. Acad. Sci. U.S.A., 66, 793 (1970). Wallach, D. F. H. and Zahler, P. H., Proc. Nat. Acad. Sci. U.S.A., 6, 1552 (1966). Steim, J. M. and Fleischer, S., Proc. Nat. Acad, Sci. U.S.A., 3, 1292 (1967). Urry, D. W., Masotti, L. and Krivacic, J. R., Biochim. Biophys. Acta, 241, 600 (1971). Kiehn, E. D. and Holland, 3. J., Proc. Nat. Acad. Sci. U.S.A., 61, 1370 (1968). 834

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16. 17. 18. 19. 20. 21. 22.

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Narchesi, S. L., Steer, E., Marchesi, V. T. and Tillack, T. W., Biochem., 2, so (1971). Berg, H. C., Biochim. Biophys. Acta, 183, 65 (1969). Lenard, J., &o&em., 9, 1129 (1970). Steck, T. L., Fairbanks, G. and Wallach, D. F. H., Biochem., lo, 2617 (1971) Dunker, A. K. and Rueckert, R. R., J. Biol. Chem., 244, 5074 (1969). Lowry, 0. H., Rosenberg, N. J., Farr, A. L. and Randall, R. J., J. BioZ. Chem., 195, 357 (1951). Tanford, C., in Physica Chemistry of MacromoZecuZes (John Wiley and Sons, N. Y., 1961).

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