Biologia Estrutural. Estrutura de proteínas - perspectiva histórica. Premio Nobel de Química

(1)

Biologia Estrutural

• Estrutura do curso

• Programa

• Estrutura de proteínas - perspectiva histórica

(2)

•

Aulas teóricas

•

Discussão de artigos

•

Tutoriais na sala multimedia

Formato

Avaliação

Equipe

• Guilherme Menegon Arantes (B9, sala 915, garantes@iq.usp.br, http://gaznevada.iq.usp.br) • Roberto K. Salinas (B10, sala 1000, roberto@iq.usp.br, www.iq.usp.br/roberto)

• Monitora: Luciana Coutinho de Oliveira (B10, sala 1004, luciana@iq.usp.br) • Professores convidados: Cristiano Oliveira (IFUSP) e Shaker C. Farah (IQUSP)

•

Seminários sobre artigos escolhidos e designados pelos professores

•

Relatório descrevendo um segundo artigo científico, escolhido pelos

(3)

Programa

www.iq.usp.br/roberto/aulas.html

Lista de artigos

(4)

VOL.

37,

1951

CHEMISTR

Y:

PA

ULING,

COREY,

BRA

NSON

by

an

increase

in

protein

content, while

the

amount

of

_{desoxyribonucleic}

acid

remains unchanged.

Acknowledgments.-This

work was

supported

by

research

_grants

fromi

the

University

of

California

Board

of Research.

We

are

greatly

indebted

to

Professor

A. W.

Pollister,

Dept.

of

Zoology,

Columbia

_University,

for

allowing

the

senior

author

use

of his

laboratory

facilities

to

conduct

the

measurements described

herein.

I

_Salvatore,

_{C. A., Biol.}

_Bull.,

_{99, 112-119 (1950).}

2 _{Caspersson, T.,}

_Skand.

_Arch.

_Physiol.,

_73,

_Suppl.

₈

_(1936).

3

Pollister,

A. W.,

and

Ris, H.,

Cold Spring

Harbor

Symp.

Quant. Biol.,

_{12, 147-157}

(1947).

4Swift,

H. _H.,

_Physiol.

_Zool.,

_23,

169-198

(1950).

Swift,

H. _H.,

these

_{PROCEEDINGS, 36,643-654}

_(1950).

6 _{Ris, H.,}

and

Mirsky, A.

E.,

J. Gen.

Physiol.,

33, 125-146

(1949).

7

_{Leuchtenberger,}

_C.,

_Vendrely,

_{R., and}

_Vendrely,

_C.,

_these

_PROCEEDINGS,

_37,

_33-37

*(1951).

8

_Alfert,

_{M., J.}

_Cell.

_Comp.

_{Physiol., 36,381-410}

_(1950).

9

Schrader,

_F.,

_and

Leuchtenberger,

C., Exp.

_Cell

_Res.,

_1,

_421-452

_(1950).

10

_{Pollister, A. W.,}

_and

_{Leuchtenberger,}

_C.,

_{these PROCEEDINGS,}

_35,

_{66-71 (1949).}

11

_{Leuchtenberger,}

_C.,

_Chromosoma,

_3,449-473

_(1950).

12 _Mirsky,

A. E.,

and

Ris,

H.,

Nature, 163, 666-667

(1949).

THE

STRUCTURE

OF PROTEINS: TWO HYDROGEN-BONDED

HELICAL

CONFIGURATIONS

OF

THE POL YPEPTIDE CHAIN

By

LINUS PAULING, ROBERT

B. COREY, AND H. R. BRANSON*

GATES

AND

CRELLIN

LABORATORIES

OF

CHEMISTRY,

CALIFORNIA

INSTITUTE

OF

TECHNOLOGY,

PASADENA,

CALIFORNIAt

Communicated

February

28,

1951

During

the

past

fifteen

years we

have been

attacking the problem

of the

structure

of

proteins

in

several

ways.

One

of

these

ways

is

the complete

and

accurate

determination

of

the crystal

structure of amino

acids,

pep-tides,

and other

simple substances related

to

proteins,

in order that

infor-mation about

interatomic

distances,

bond angles,

and

other configurational

parameters

might

be

obtained

that

would permit

the

reliable

prediction of

reasonable

configurations

for

the

polypeptide

chain.

We

have now

used

this

information

to

construct

two

reasonable

_{hydrogen-bonded}

helical

con-figurations

for the

_{polypeptide chain;}

wte think

that

it is

likely

that

these

configurations

constitute

an

_important

part

of the structure of both fibrous

and

globular proteins,

as

well

as

of

synthetic polypeptides.

A

letter

an-nouncing

their

discovery

was

published

last

year.

'

The

problem

that

we

have

set

ourselves

is

that

of

finding all

hydrogen-bonded

structures

for

a

single

polypeptide

chain,

in

which the residues

are

205

Pauling, Corey e Branson (1951) PNAS 37:205-211

(5)

Estrutura de proteínas - princípios

Pauling, Corey e Branson (1951) PNAS 37:205-211

CHEMISTRY: PA ULING, COREY, BRANSON PROC. N. A. S.

equivalent (except for the differences in the side chain R). An amino acid

residue (other than glycine) has no symmetry elements. The general

oper-ation of conversion of one residue of a single chain into a second residue

equivalent to the first is accordingly a rotation about an axis accompanied by translation along the axis. Hence the only configurations for a chain

compatible with our postulate of equivalence of the residues are helical configurations. For rotational angle 1800 the helical configurations may

degenerate to a simple chain with all of the principal atoms, C, C' (the

carbonylcarbon), N, and 0, in thesameplane.

We assume that, because of the resonance of the double bond between

the carbon-oxygen and carbon-nitrogen positions, the configuration of each

residue >N-C6 is planar. This structural feature has been

verified for each of the amides that

IZi.23 _we _have _{studied. Moreover, the}

resonance theory is now so well CV/ grounded and its experimental

sub-1o stantiation so extensive that there H N 120° can be no doubt whatever about its

120O _application to the amide group.

The observed C-N distance, 1.32

io

C° H

R

iA,

_corresponds _to _nearly _{50 per cent}

double-bond character, and we may

conclude that rotation by as much

0

as

100

from the planar configuration

would result in instability by about 1 kcal. mole-'. The interatomic

N _H _distances _and _bond _{angles within} the residue are assumed to have the

values shown in figure 1. These (j+Hc values have been formulated2 by

consideration of the experimental

values found in thecrystal structure FIGURE 1 studies of DL-alanine,3 L-threonine,4

Dimensions of _{the polypeptide} chain. _{N-acetylglycine5,} and ,-glycylgly-cine6 that have been made in our

Laboratories. It is further assumed that each _nitrogen atom forms a

hy-drogen bond with an _oxygen atom of another residue, with the

nitrogen-oxygen distance equal to 2.72

A,

and that the vector from thenitrogen atom

to the hydrogen-bonded oxygen atom lies not more than 300 from the N-H

direction. The energy of an N-H

-* - 0=Chydrogen bond is of the order

206

1)Teoria sobre estruturas de ressonância

2)Dados de cristalografia de pequenas moléculas

(6)

VOL. 37, 1951 CHEMISTR Y: PAULING, COREY,BRANSON

FIGURE 2

The helix:with3.7residuesperturn.

FIGURE 3

Thehelix with 5.1residues per turn.

207

hélice-alfa hélice-gama

Erro: Pauling desenhou uma hélice que gira para a esquerda, assumindo aminoácidos

com configuração D ao contrário de L. A alfa-hélice regular gira para a direita, os

(7)

Um método semelhante foi utilizado por Watson e Crick

para propor a estrutura do DNA

(8)

Pauling e Corey propuseram também um outro tipo de estrutura

secundária: a folha-beta pregueada

paralela

anti-paralela

CHEMISTR Y: PA ULING AND COREY

acid residues are related differently to the structure when attached to one a carbon atom than when attached to the a carbon atom of an adjacent residue. The pleated-sheet configuration can accordingly be described

as involving only one kind of glycine residue, in case that it were to be

as-sumed by a polyglycine, but two kinds of residues for all optically active

amino-acid polymers. These two kinds differ in that, for the L configura-tion, a residue of one kind points its ,(carbon atom in the C=O direction,

and a residue of the other kind points its ,B carbon atom in the N-H direc-tion.

We have found some evidence to support the belief thatthe pleated-sheet configuration is present in stretched muscle, stretched hair, feather

kera-tin, and some other fibrous proteins that have been assigned the (3-keratin

structure. These proteins give x-ray diagrams on which there is a strong

meridional

reflection

corresponding to spacing about 3.3 A, which is a few per cent larger than the fiber-axis distance per residue for the undistorted

TABLE 1

COORDINATES OF ATOMS IN THE POLYPEPTIDE PLEATED-SHEET CONFIGURATION (IN A)

- UNROTATED--- , 7° ROTATION - 20 ROTATION-.

ATOM X y S X y 5 X y S C1 0.00 1.15 0.00 0.00 1.09 0.00 0.00 0.96 0.00 N1 -0.36 0.30 1.14 -0.36 0.46 1.17 -0.36 0.35 1.29 C1' 0.53 -0.28 1.91 0.53 -0.31 1.96 0.50 -0.40 1.98 01 1.74 -0.14 1.73 1.72 -0.31 1.75 1.63 -0.64 1.58 C2 0.00 -1.15 3.07 0.00 -1.09 3.15 0.00 -0.96 3.32 N2 -0.36 -0.30 4.21 -0.36 -0.39 4.31 -0.34 -0.14 4.49 C2' 0.53 0.28 4.98 0.53 0.22 5.12 0.50 0.08 5.49 02 1.74 0.14 4.80 1.72 -0.04 4.95 1.63 -0.39 5.50 C1* 0.00 1.15 6.14 0.00 1.09 6.30 0.00 0.96 6.64

pleated sheet, but much smaller than the value 3.6 A for fully extended

polypeptide

chains. We have noticed that the pleated sheet can be

sub-jected, without rupturing the hydrogen bonds, to a considerable

distor-tion, in such a way as to increase the fiber-axis distance. This distortion is

effected

by

rotating each amide group about its

C-C*

axis through a small

angle.

The rotation moves one of the two ,B positions of each carbon atom

farther from the median

plane

and the other nearer, and the effective

rota-tions for the two

_{non-equivalent}

_{kinds of} _optically _active _residues are such

as to permit each to be an L residue with its side chain farther from the

median

plane

than in the undistorted structure.

Presumably

the van der

Waals

repulsion

of the side chain atoms and the main chain atoms would

be operating in proteins of normal chemical composition with the pleated-sheet

configuration,

and this would cause some distortion of the

chain-lengthening sort.

(It

is to be noted that two kinds of pleated sheets can be constructed of L-amino-acid residues, of which for one the deformation that

VOL. 37, 1951 253

CHEMISTR Y: PA ULING AND COREY

acid residues are related differently to the structure when attached to one a carbon atom than when attached to the a carbon atom of an adjacent residue. The pleated-sheet configuration can accordingly be described

as involving only one kind of glycine residue, in case that it were to be

as-sumed by a polyglycine, but two kinds of residues for all optically active

amino-acid polymers. These two kinds differ in that, for the L configura-tion, a residue of one kind points its ,(carbon atom in the C=O direction,

and a residue of the other kind points its ,B carbon atom in the N-H direc-tion.

We have found some evidence to support the belief that the pleated-sheet configuration is present in stretched muscle, stretched hair, feather

kera-tin, and some other fibrous proteins that have been assigned the (3-keratin

structure. These proteins give x-ray diagrams on which there is a strong

meridional

reflection

corresponding to spacing about 3.3 A, which is a few per cent larger than the fiber-axis distance per residue for the undistorted

TABLE 1

COORDINATES OF ATOMS IN THE POLYPEPTIDE PLEATED-SHEET CONFIGURATION (IN A)

- UNROTATED--- , 7° ROTATION - 20 ROTATION-.

ATOM X y S X _y 5 X _y S C1 0.00 1.15 0.00 0.00 1.09 0.00 0.00 0.96 0.00 N1 -0.36 0.30 1.14 -0.36 0.46 1.17 -0.36 0.35 1.29 C1' 0.53 -0.28 1.91 0.53 -0.31 1.96 0.50 -0.40 1.98 01 1.74 -0.14 1.73 1.72 -0.31 1.75 1.63 -0.64 1.58 C2 0.00 -1.15 3.07 0.00 -1.09 3.15 0.00 -0.96 3.32 N2 -0.36 -0.30 4.21 -0.36 -0.39 4.31 -0.34 -0.14 4.49 C2' 0.53 0.28 4.98 0.53 0.22 5.12 0.50 0.08 5.49 02 1.74 0.14 4.80 1.72 -0.04 4.95 1.63 -0.39 5.50 C1* 0.00 1.15 6.14 0.00 1.09 6.30 0.00 0.96 6.64

pleated sheet, but much smaller than the value 3.6 A for fully extended

polypeptide

chains. We have noticed that the pleated sheet can be

sub-jected, without rupturing the hydrogen bonds, to a considerable

distor-tion, in such a way as to increase the fiber-axis distance. This distortion is

effected

by

rotating each amide group about its

C-C*

axis through a small

angle.

The rotation moves one of the two ,B positions of each carbon atom

farther from the median

plane

and the other nearer, and the effective

rota-tions for the two

non-equivalent

kinds of optically active residues are such

as to permit each to be an L residue with its side chain farther from the

median

plane

than in the undistorted structure.

Presumably

the van der

Waals

repulsion

of the side chain atoms and the main chain atoms would

be operating in proteins of normal chemical composition with the pleated-sheet

configuration,

and this would cause some distortion of the

chain-lengthening sort.

(It

is to be noted that two kinds of pleated sheets can be constructed of L-amino-acid residues, of which for one the deformation that

(9)

Stereochemistry of polypeptide chain configurations (1963) J. Mol. Biol. 7, 95-99 Aula 1 - Introdução/ estrutura de proteínas/PDB 1

Gráfico de Ramachandran

(10)

Estrutura cristalográfica da mioglobina a 2Å de resolução

J.C. Kendrew et al. (1960) Nature 185: 422-427

- Kendrew confirmou as observações feitas por Perutz com um mapa de 6 Å"

- Determinou de forma inequívoca o traçado da cadeia, incluindo o N- e C-terminal"

- Confirmou que a estrutura da hélice-alfa de Pauling encaixava-se bem no mapa de densidade eletrônica, e que a direção da hélice-alfa era

para a direita

(11)

Estrutura da lisozima de clara de ovo de galinha a 2Å

PDB 1LYZ

-primeira confirmação da existência das folhas-beta pregueadas propostas por Pauling -Ao contrário da conformação proposta por Pauling e Corey, folhas-beta são retorcidas

(12)

Três ângulos diedrais (Φ, Ψ e ω) são suficientes para descrever a

conformação do esqueleto polipeptídico

(13)

δ+

(14)

(15)

Hairpins, fitas-beta paralelas e antiparalelas

hairpin

paralela

(16)

(17)

(18)

Twist

Diagramas de topologia ajudam a compreender a estrutura

Folhas-beta

(19)

Alfa-Beta

Beta

Alfa

Alfa-hélices são comumente

utilizadas para formar folhas-beta

(20)

(21)

Qual a relação entre similaridade de sequência e de

estrutura terciária?

•

homólogos: duas proteínas são homólogas quando derivam do mesmo precursor, e

portanto são semelhantes em estrutura mas não necessariamente em função

•

identidade: quando 50% dos aminoácidos de duas proteínas são iguais elas

possuem 50% de identidade e não 50% de homologia, elas são homólogas

The EMBO Journal vol.5 no.4 pp.823-826, 1986

The

relation between the divergence

of sequence and structure

in

proteins

Cyrus Chothial and Arthur M.Lesk2

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, and 'Christopher Ingold Laboratory, University College London, 20 Gordon Street, London WC1H OAJ, UK

2Permanent address: Fairleigh Dickinson University, Teaneck-Hackensack Campus, Teaneck, NJ 07666, USA

Communicated by M.F.Perutz

Homologous proteins have regionswhich retain the same

gen-eral fold and regions where the folds differ. For pairs of

distantly related proteins (residue identity --20%), the regions

with the same fold may comprise less than half ofeach mol-ecule. The regions with thesame general fold differ in

struc-ture by amounts that increase as the amino acid sequences

diverge. The root mean square deviation in the positions of

the main chain atoms, A, is relatedto the fraction of mutated residues, H, by the expression: A(A) = 0.40 el87H.

Key words: evolution/protein homology/model building

Introduction

The comparative analysis of the structures of related proteins can

reveal the effects of the amino acid sequence changes that have occurred during evolution (Perutz et al., 1965). Previous work

on individual protein families has shown thatmutations, insertions

and deletions produce changes in three-dimensional structure

(Almassy and Dickerson, 1978; Lesk and Chothia, 1980, 1982,

1986; Greer, 1981; Chothia and Lesk, 1982, 1984; Read etal.,

1984). Here we report a systematic comparisonofstructures from

eight different protein families. This shows that the extent of the structural changes is directly relatedto the extent ofthe sequence

changes.

In the work reported here we used the atomic coordinates of 25 proteins (Table I). All these structures have been determined

at high resolution (1.4-2.OA) and refined. The errors in their co-ordinates are 0.15-0.20A (see references given in Table I). The 25 proteins represent eight different protein families and

pro-vide 32 pairs of homologous structures.

Methods and Results

The conserved structural coresand the variable regions of

hom-ologous proteins

The structures ofhomologous proteins can be divided into those

regions in which the general fold of the polypeptide chains is

very similar and those where it is quite different. In comparing protein structures it is useful to separate the parts that have similar folds from those where the folds differ. We did this using the

following quantitative procedure: (i) the main-chain atoms of

major elements ofsecondary structure - helices or two adjacent

strands of 3-sheet - were individually superposed; and (ii) each superposition was then extended to include additional atoms at

both ends. The extension was continued as long as the deviations

in the positions of the atoms in the last residue included were

no greater than 3 A. This procedure defined the segments that

1-0 oL.c 0 u 0 E E uQU ₀₄ ._.6 -o In w '6-t02 ix04 a1.. 0 I-0~ 10-100 80 60 Sequence 40 Identity

(0/)

20 0

Fig. 1. Size of common cores as a function of protein homology. If two

proteins of length n1 and n2 have c residues in the common core, the

fractions of each sequence in the common core are c/n1 and c/n2. We plot

these values, connected by a bar,- against the residue identity of the core

(see Table II).

oi._ £ 0 ._ o a V L-o a. C 0 0 0 100 80 60 40 20 0

Percent residue identity

Fig. 2. The relation of residue identity and the r.m.s. deviation of the

backbone atoms of the common cores of 32 pairs of homologous proteins

(see Table I).

823 l I

J

|

II

|

K,I

I I

IF

0.

^

(22)

(23)

Formato do arquivo PDB

x

y

z

Nome do átomo Número do

aminoácido

(24)

x

y

z

(x

1 ,y

1 ,z

1 )

(x

2 ,y

2 ,z

2 )

Cα

_Cα

Rotações e translações

(x

1 ,y

1 ,z

1 )’

(x

2 ,y

2 ,z

2 )’

…

(x

n

,y

n

,z

n

)’

= R

(x

1 ,y

1 ,z

1 )

(x

2 ,y

2 ,z

2 )

…

(x

n

,y

n

,z

n

)

(25)

Como quantificar a similaridade/diferença entre duas

estruturas?

(26)

RMSD (Root Mean Square of the Deviations)

Desvio padrão médio das diferenças das posições

x

y

z

Cα

r

0 r-r

0

€ RMSD = 1 N ! r − r ! ₀

(

)

2 i=1 N

∑

= 1 N

(

x − x0

)

2 + y − y

(

₀

)

2 + z − z

(

₀

)

2 i=1 N

∑

!

€

α ≡ lim

_{N $ →}_{$ ∞}

1 N

∑

x

i

−

µ

)

*

+

,

-

. !

!

€

σ

2

= lim

_{N # →}_{# ∞}

1 N

(

x

i

−

µ

)

2

∑

(

)

*

+

_,

- !

desvio médio

variança

Sobreposição de duas estruturas envolve encontrar a orientação

tal que o RMSD entre elas seja mínimo

x

pr

obab.

(27)