Download pdf - Properties of nucleation sites in globular proteins

Vol. 97, No. 4, 1980

December 31, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1582-1590

PROPERTIES OF NUCLEATION ~ITES IN GLOBULAR PROTEINS

P.K.PON~SWAMY and M.PRAEHAKA~AN

Department of Physics, Autonomous Postgraduate Centre,

University of Madras, Tiruchirapalli 620020,

Tamilnadu, INDIAo

Received October 15,1980

S~,~4.~Y: Using crystal data for a set of proteins and hydro-

phobic indices for amino acids, a criterion is dra~m to identify

nucleation sites in globular proteins. The properties of these

sites are described in terms of certain relevant parameters and

a suggestion is made on the growth and aggregation of nucleation

sites in protein molecules°

A nascent polypeptide chain folds very quickly to form

the active-native structure of the protein, indicating the

possibility of the existence of a few preferred folding pathways

o~t of the innumerable probable conformational states. The

formation of nucleation sites along the sequence of the poly-

peptide due to local interactions is considered to be one of the

possible ways to make a selection of pathways and on which model,

appealing reports are now appearing° From the observed tertiary

patterns of a few globular protein cz~ystals, Wetlaufer (1) first

suggested the possibility of the formation of nucleation regions

by applying the rule of continuous segments in amino acid

sequence, and analysed the u in detail. Matheson and Scheraga (2)

proposed nucleation pockets from the knowledge of short- and

medium-range interactions in proteins. Kanehisa and Tsong (3)

conceived fluctuating local clusters in native structure forma-

This work was supported by a grant by the Department of ~cience and Technology, Government of India to PE~.

0006-291 X/80/241582-09501,00/0 Copyright © 1980 by Academtc Press, Inc. A ll rights o f reproductton in any form reserved. 1582

Vol. 97, No. 4, 1980 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tion and described the self assembly kinetics. Rose and Roy (4)

suggested that chain sites corresponding to local maxima in

hydrophobicity serve as folding primitives. Recently from the

laboratory of the present authors, a report was made (5) on the

possibility of predicting nucleation sites by the application of

the concept, called the 'surrounding hydrophobicity' for a

residue in the protein matrix. The decisive role played by hydro-

phobic bonding through long-range interactions could be well

realised by the observance of very stable hydrophobic domains in

almost all the known crystal structures of globular protein mole-

cules. In this communication we report the results of our recent

study on the characteristics of nucleation sites in fourteen

proteins.

~H0~

As the hydrophobic clustering is conceived to be one of

the important features in the formation of globular structures in

proteins, we studied the concept of nucleation domains by utilising

the hydrophobic indices of aaino acid residues as given by Tanford (6)

and by Jones (7). Using these experimental quantities we define

what is termed the 'surrounding hydrophobicity' for a residue in

a protein molecule in its crystalline fora: The auino acid residues

are represented by their o-carbon (crystal) coordinates and each

of the residues is assigned with the Tanford-Jones index. The

surrounding hydrophobicity of a residue in the protein matrix

is defined as the sum of the hydrophobic indices of various

residues appearing ~thin 8 A radius limit from the residue in

question (see ref. (8) for details). This bulk index measures

the effective hydrophobic bonding made by the residue with other

interacting residues in the molecule. The residues having

surrounding hydrophobicity values equal to or greater than twice

1583


the average value for all the residues in the protein are here

assumed to form 'hydrophobic domains' and the residue of the

highest surrounding hydrophobicity within a domain is taken to

represent the site of that domain. The site identified in this

way in a protein could then taken to be the result of the atomic

interactions (short-, medium- and long-range) within the effective

range of surroundings in that protein.

The probable nucleating portions of a protein molecule

arising from short- and medium-range interactions alone were

determined as follows: the hydrophobic indices of four re3idues on

either side of a residue were added to that residue and this was

repeated along the length of the protein chain; the residues

having hi~est sumued hydrophobic indices were then identified and

if they were noted to fall within any of the nucleation domains

predicted from considerations of total interactions, they are

assumed to be the nucleation sites arising from interactions

devoid of long-range nature.

For purposes of determining the spatial positions of the

nucleation sites, the protein is assumed to be an ellipsoid of

semi-axes a,b and c, whose volume is just sufficient to enclose

all the residues in it; these medium-sized ellipsoids were

determined by an iterative procedure involving rotations and

translations about the coordinate axes fixed on the centroid

of the protein crystal as described in one of our earlier

reports (9). The spatial position of a residue i with reference

to the centroid of the ellipsoid is given by

2 2 Z~

d i = a2 ~

where xi, Yi and z i are the Cartesian coordinates of the

residue i, the origin coinciding the protein centroid; d i varies

1584

VOI. 97, No. 4, 1980 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from 0 to l, the zero value corresponding to the aost interior

point and the unity value corresponding to the most exterior or

surface point of the protein.

The accessible contact areas for a solvent molecule of

1o4 A radius were computed for each of the residues in the

protein by the method of Lee and Richards (10).

The maintanance of the native folded structure in a

protein molecule is mainly due to Van der Waals interactions

and hence the stability contribution from these interactions

was determined for each residue from all other residues by

applying the Lennord Jones function and parameters developed

by Scheraga's group. 2~ne segments of the protein chain which

are highly stabilised by Van der Waals interactions were then

identified and those containing any of the nucleating domains

determined from total interactions were taken to represent

Van der Waals ener~ dominating segments.

RESULT3 AI~D DIbCUS~ION

The para~ueters defined above and determined for 14

different globular proteins are given in Table l; the secondary

structures associated with the nucleating domains and the sizes

of the domains in terms of the number of associated residues are

also given in this Table. An analysis of this Table reveals

many characteristic properties of nucleation sites in globular

proteins.

We note at least two nucleation sites for a protein, the

bigger on~s having up to five sites. ~Iost of the site residues

are of nonpolar type, but the occurrence of residues ~ith polar

atoms in their sidechains, viz., Set, T%~r, Tyr, Arg, Gln, Asn,

1585


.H

4J 0

P~

,-4

m ©

-H

H %

ID 4a .el

.H -p

O

O 0 ,

m ~

g

~ o o

~c~ ..Pew

c 'd~J

• r'l ~ 4 " I : " d ~ Ph--'t

~)

"~ 0 0

0 o .H r-I h ~ o c ~ h.'-d 0

o

I I

~ ) ~ . 1 ~

~ ) . ~ r- I . r l

0 I

P.I

~ ~ ~ o o ~, ~ ~ P o L~

Ch ~G I ~

0 ~ 0 ~ ~

I I I I I I H r - t H - H " ~ H

r-I H H

t~0 --,~- G', b'~, kD Cq C'] 0 0 L G b ~ , ~ ' P - ~ r ~ " l O b , P ' - ' D LO

0 i ~ l ~ l l c,,i b'l HI o]., 'II l ' I HI " l kO ~-I 0 ['c',( ~ ~'- ,--I kO ,'D C-" "-," tr~ 0 I:~" h% ~ t C (%1 ,~J C , C,I r -~r" "Y

O['-- ~ ~ h'~ ~'~ H 0 03 0 cJ ~'- : L?, "~, o: C" O :'u ' " CC, --~

0J I N H H H o,l I ( \ ] 0 -~ ~I t r~CX] I H H f ~ H C J , ' I I H I I I I 1 I I I I I I I I I I I I I

~ ~ o o o o o ~ o o ~ o o o o o o o , ~ ~ ~ ~0 O0 OLd, O~ 0 0 0 ~ ~ 0 ~ 0 O0 ~ . . . . . . . . . . . . . . . . . . d d J d O ~ ~ 0 O 0 O 0 0 ~ 0 0 0 0 0 0 ~ 0

O 0 O 0 O 0 O 0 O 0 0 0 0 0 0 ~ 0 0 O 0 O 0

kO K", O 0 O 0 O 0 O 0 0 0 0 0 0 0 0 0 O 0 CO ":t" . - I r'"l 0 t " ' 0 4 0 ',D "~" kO U". "d" "@ 0 -~ 0 cO OhCJ CO0-~ G', C,.

(~.~ (X,l C'~04 (",J Ck~ (kJCM 0 4 N OJ 040 . J ©d C'4 04 N C,. I OJC'J CM .,",J

Z ~ -~ ~ - I l~ ~I ~ o:~,'~.oD_ ~ PQ.~ •

.H

I 0

0 4 0 ~ ~ ~ ~ ~ ~ ~0 O ~ ~ ~ 0 O ~ ~ ~ 0 ~ ~ 0 4 ~ 0 4 0 0 4 ~

. ~ H . ~I o I

• . P I~1 ~ ~ o ~1 I ~1 ® - P O o I:~ r~ o ~ o I o ~ o ~ I.i i - I .H I - - I ~ • ~ 1 ~ I . - I . H

o o • ,4 ~1 -,-I ~1 I ~ ~1 -P ~ I

"t~ -tl l:l

1586


0 0 v

I--I

• ,-t .q ,q ~ . - ~ ~ o

(D ~ . H O

c3 P.

O 0 ~ m

n ~ . ?

e' ':.:.3 ~ e

I0.:, ~ - -~ .....

I 4 ~ ~ ,--i

~ b ' ~ "m ~ 04O' . ~ ~ 12"- 0

L, ~,

I I I I I I I I O f " 0 3 0 0 "~- OJ C) t¢'~ t""- 0 .~: 0"~ r'-I <'. d 4 ( : 0 0 0 0 00 t,~. ~'~ 0 " ~ 0

00 r.-I

O C , O 0 0 0 0 oJ 0 ' ~ 0 L~ ~r'~ [,r~ ! r-{ 0 I ~ L O 00 LO L~, ,--I .~-LO r-I 0 LC~',-D

o o ~

.-~ ~.~

~I --~

4

o o .N f-I ~ o ~

o

o ~1

I I

~,'.! ,'el ~ 4 ~

% 4 . , - t

~)

0

' ~ ~ ~ b ~ 0 0 ~ 0 0 0 ~ ~ ~ 0 0 O O t ~ O O ~ b ~,

4 0 O 0 ~ 0 0 0 0 0 0 0 ~ 0

~ 0 0 0 ~ 0 ~ ~ ~

O O O 0 0 0 0 0 0 0 0 0 0 0

O O O 0 0 0 0 0 0 0 0 0 0 0

O 0 ~ ~ ~ ~ D ~

~ ~ ~ 0 ~ ~ 0 Od ,"-I ~

I - ~

0 o

4

r---I

r'q

.,-q

c-

4~

d 0

o ~4

.~.~

o

o

o

o o ..~

o 4 o

0:, 0

o

0

.,-I

4 ~ 4 ~ .~ . r t

-el

%

4 ~ ~'1

~'1 I0

®

1587


. Q ~ I 4 • 74/~.~

Thermolysin

Lysozyme oC- Chymotrypsin

i ~ . 1.

tlO/4Z :~

$ubtilisin BPN'

Concanavalin A

JiJooJitions of nucleation sites in 71obuler

proteins. The diste~ce (in A units) bet~een sites

are $ivez~ near the connectin~ lines ~nd the existing

hydrophobic channels are marked by curved dashed

lines. Aaino acid sequence numbers for the site

residues are ~-iven inside the circles. Proteins

not shown in this Fig. have only two nucleation

sites each and hence left out.

and His is also to be noted. The nucleation sites in a protein

(e~cept in one case) are seoarated by at least 20 residues along

the sequence. The size of each domain varies from 13 to 20

residues, many having over 15 residues, the optimum number

suggested by Uetlaufer (1). The positions of various nucleation

sites in each of the proteins are indicated in Fi~. 1. Interes-

tingly, in many of the proteins, a few residues belonging to one

nucleation domain also become members of another domain thereby

interconnecting them; this feature indicates the possibility of

1588


existing a long hydrophobic core or channel across the protein

matrix; such channels noted in the presently studied proteins

are marked by dashed lines in Fig. 1.

Invariably, the domains are well buried and dens!y packed

in the protein matrix, the representative site residues having

nearly zero accessibilities. By their nature, the polar-nucleating

site residues retain small contact areas for solvent interaction.

The nucleation site residues acquire surrounding hydrophobicities

(hydrcphobic bonding energy, so to say) around 20 to 29 kcal/mole

and Van der Waals stabilization around 8 to 23 kcal/mole. Interes-

tingly, the peptide units that acauire some amount of nucleating

property from short- and medium-range interactions alone become

parts of seguents that are able to receive the best stability

from Van der Waals interactions, and consequently, fall within the

nucleation domains predicted from considerations of total interac-

tions. The most interesting observation is that, although alpha-

helical structure is also associated with the nucleating domains

(sites) the p-strand is the dominating secondary structure in

them. This result is in accordance with our earlier prediction (5)

tl~t the p-sheets are the most buried parts in globular proteins.

These characteristic results on nucleation domains/sites

provide support to the following important concept in the problem

of protein folding: 'the short- and medium-range interactions from

the primary sequence provide the minimum force to create nucleation

sites, and the long-range Van der Waals/hydrophobic interactions

provide the environment for their growth and aggregation, and

stability against disturbing external forces and conformational

fluctuations'.

1589


REFERENCES

1. Wetlaufer, D.B. (1973) Proc. Natl. Acad. Sci. USA 70,

697-701.

2. Matheson, R.R. and Scheraga, H.A. (1978) Macromolecules,

ll, 819-8 29.

3. Kanehisa, M.I. and Tsong, T.Y. (1979) Biopolymers, 18

1375-78, 2913-2928.

4. Rose, G.D. and. Roy, S. (1980) Proco iffatl. Acad.. Jci. USA

77, 4643-4647.

5. Ponnuswamy, P.K., Prabhakaran, M. and Manawalan, P. (1980)

Biochim. Biophys. Acta 62_~3, 301-316.

6. Tanford, C. (1963) J. Am. Chem. Joc. 84, 4240-4247.

7. Jones, D.D. (1975) J. Theor. Biol. 50, 167-183.

8. Manavalan, P. and. Po~_nuswaray, P.K. (1978) Nature 27~,

673-674.

9. Prabhakaran, M. and. Ponnuswauy, P.K. (1980)

(In press).

10.

J. Theor. Biol.

Lee, B. and Richards, Folio (1972) J..~Io!. Biol. 55,

1590