Encyclopedia of Biological Chemistry - Vol_4.pdf - med - pet300

Secondary Structure in

Protein Analysis

George D. Rose

The Johns Hopkins University, Baltimore, Maryland, USA

D EGREES OF F REEDOM

IN THE B ACKBONE

The six backbone atoms in the peptide unit [Ca( i ) – CO –

NH – Ca( i þ 1)] are approximately coplanar, leaving

only two primary degrees of freedom for each residue.

By convention, these two dihedral angles are called f

and c( Figure 2 ). The protein’s backbone conformation

is described by the f,c-speciﬁcation for each residue.

Proteins are linear, unbranched polymers of the 20 naturally

occurring amino acid residues. Under physiological conditions,

most proteins self-assemble into a unique, biologically relevant

structure: the native fold. This structure can be dissected into

chemically recognizable, topologically simple elements of

secondary structure: a-helix, 3 10 -helix, b-strand, polyproline

II helix, turns, and V-loops. Together, these six familiar motifs

account for ,95% of the total protein structure, and they are

utilized repeatedly in mix-and-match patterns, giving rise to the

repertoire of known folds. In principle, a protein’s three-

dimensional structure is predictable from its amino acid

sequence, but this problem remains unsolved. A related, but

ostensibly simpler, problem is to predict a protein’s secondary

structure elements from its sequence.

C LASSIFICATION OF S TRUCTURE

Protein structure is usually classiﬁed into primary,

secondary, and tertiary structure. “Primary structure”

corresponds to the covalently connected sequence of

amino acid residues. “Secondary structure” corresponds

to the backbone structure, with particular emphasis on

hydrogen bonds. And “tertiary structure” corresponds

to the complete atomic positions for the protein.

Protein Architecture

A protein is a polymerized chain of amino acid residues,

each joined to the next via a peptide bond. The

backbone of this polymer describes a complex path

through three-dimensional space called the “native

fold” or “protein fold.”

Secondary Structure

Protein secondary structure can be subdivided into

repetitive and nonrepetitive, depending upon whether

the backbone dihedral angles assume repeating values.

There are three major elements (a-helix, b-strand, and

polyproline II helix) and one minor element (3 10 -helix)

of repetitive secondary structure ( Figure 3 ). There are

two major elements of nonrepetitive secondary structure

(turns and V- loops).

C OVALENT S TRUCTURE

Amino acids have both backbone and side chain

atoms. Backbone atoms are common to all amino

acids, while side chain atoms differ among the 20

types.

Chemically,

amino

acid

consists

central, tetrahedral carbon atom, (

), linked cova-

R EPETITIVE S ECONDARY S TRUCTURE:

THE a-H ELIX

When backbone dihedral angles are assigned repeating

f,c-values near (2608, 2408), the chain twists into a

right-handed helix, with 3.6 residues per helical turn.

First proposed as a model by Pauling, Corey, and

Branson in 1951, the existence of this famous structure

was experimentally conﬁrmed almost immediately by

lently to (1) an amino group ( – NH 2 ), (2) a carboxyl

group ( – COOH), (3) a hydrogen atom ( – H) and (4)

the side chain ( – R). Upon polymerization, the amino

group loses an – H and the carboxy group loses an

– OH; the remaining chemical moiety is called an

“amino acid residue” or, simply, a “residue.” Resi-

dues in this polymer are linked via peptide bonds,

as shown in Figure 1 .

SECONDARY STRUCTURE IN PROTEIN ANALYSIS

FIGURE 1 (A) A generic amino acid. Each of the 20 naturally occurring amino acids has both backbone atoms (within the shaded rectangle) and

side chain atoms (designated R). Backbone atoms are common to all amino acids, while side chain atoms differ among the 20 types. Chemically, an

amino acid consists of a tetrahedral carbon atom ( – C– ), linked covalently to (1) an amino group ( – NH 2 ), (2) a carboxyl group ( – COOH), (3) a

hydrogen atom ( – H), and (4) the side chain ( – R). (B) Amino acid polymerization. The a-amino group of one amino acid condenses with the

a-carboxylate of another, releasing a water molecule. The newly formed amide bond is called a peptide bond and the repeating unit is a residue . The

two chain ends have a free a-amino group and a free a-carboxylate group and are designated the amino-terminal (or N-terminal) and the carboxy-

terminal (or C-terminal) ends, respectively. The peptide unit consists of the six shaded atoms (Ca–CO–NH–Ca), three on either side of the peptide

bond.

Perutz in ongoing crystallographic studies, well before

elucidation of the ﬁrst protein structure.

In an a-helix, each backbone N – H forms a hydrogen

bond with the backbone carbonyl oxygen situated four

residues away in the linear sequence chain (toward the

N-terminus): N – H( i )· · ·OyC( i 2 4). The two sequen-

tially distant hydrogen-bonded groups are brought into

spatial proximity by conferring a helical twist upon

the chain. This results in a rod-like structure, with the

hydrogen bonds oriented approximately parallel to

the long axis of the helix.

In globular proteins, the average length of an a-helix

is 12 residues. Typically, helices are found on the outside

of the protein, with a hydrophilic face oriented toward

the surrounding aqueous solvent and a hydrophobic face

oriented toward the protein interior.

Inescapably, end effects deprive the ﬁrst four amide

hydrogens and last four carbonyl oxygens of

Pauling-type, intra-helical hydrogen bond partners.

The special hydrogen-bonding motifs that can provide

partners for these otherwise unsatisﬁed groups are

known as “helix caps.”

In globular proteins, helices account for ,25% of

the structure on average, but this number varies.

Some proteins, like myoglobin, are predominantly

helical, while others, like plastocyanin, lack helices

altogether.

R EPETITIVE S ECONDARY S TRUCTURE :

THE 3 10 -H ELIX

When backbone dihedral angles are assigned repeating

f,c-values near (2508, 2308), the chain twists into a

right-handed helix. By convention, this helix is named

using formal nomenclature: 3 10 designates three residues

per helical turn and 10 atoms in the hydrogen bonded

ring between each N – H donor and its CyO acceptor.

(In this nomenclature, the a-helix would be called a

3.6 13 helix.)

Single turns of 3 10 helix are common and closely

resemble a type of b-turn (see below). Often, a-helices

terminate in a turn of 3 10 helix. Longer 3 10 helices are

sterically strained and much less common.

SECONDARY STRUCTURE IN PROTEIN ANALYSIS

FIGURE 3 A contoured Ramachandran (f ; c) plot. Backbone f,c-

angles were extracted from 1042 protein subunits of known structure.

Only nonglycine residues are shown. Contours were drawn in popu-

lation intervals of 10% and are indicated by the ten colors (in rainbow

order). The most densely populated regions are colored red. Three

heavily populated regions are apparent, each near one of the

major elements of repetitive secondary structure: a-helix (,2608,

2408), b-strand (,21208, 1208), P II helix (,2708, 1408). Adapted from

Hovm ¨ ller, S., Zhou, T., and Ohlson, T. (2002). Conformation of amino

acids in proteins. Acta Cryst. D58, 768 – 776, with permission of IUCr.

FIGURE 2 (A) Deﬁnition of a dihedral angle. In the diagram, the

dihedral angle, u, measures the rotation of line segment CD with respect

to line segment AB, where A, B, C, and D correspond to the x,y,z-

positions of four atoms. (uis calculated as the scalar angle between the

two normals to planes A – B – C and B – C – D.) By convention, clockwise

rotation is positive and u¼ 08 when A and D are eclipsed. (B) Degrees of

freedom in the protein backbone. The peptide bond (C 0 – N) has partial

double bond character, so that the six atoms, Ca( i ) – CO– Ca( i þ 1), are

approximately co-planar. Consequently, only two primary degrees of

freedom are available for each residue. By convention, these two

dihedral angles are called fand c 0 fis speciﬁed by the four atoms C 0 ( i )–

N–Ca–C 0 ( i þ 1) and c by the four atoms N( i )–Ca–C 0 –N( i þ 1).

When the chain is fully extended, as depicted here, f¼ c¼ 1808 :

Two b-strands in a b-sheet are classiﬁed as either

parallel or anti-parallel, depending upon whether their

mutual N- to C-terminal orientation is the same or

opposite, respectively.

In globular proteins, b-sheet accounts for about 15%

of the structure on an average, but, like helices, this

number varies considerably. Some proteins are pre-

dominantly sheet while others lack sheet altogether.

R EPETITIVE S ECONDARY S TRUCTURE :

THE b-S TRAND

When backbone dihedral angles are assigned repeating

f,c-values near (21208, 2 1208), the chain adopts

an extended conformation called a b-strand. Two or

more b-strands, aligned so as to form inter-strand

hydrogen bonds, are called a b-sheet. A b-sheet of just

two hydrogen-bonded b-strands interconnected by a

tight turn is called a b-hairpin. The average length of a

single b-strand is seven residues.

The classical deﬁnition of secondary structure found

in most textbooks is limited to hydrogen-bonded back-

bone structure and, strictly speaking, would not include

a b-strand, only a b-sheet. However, the b-sheet is

tertiary structure, not secondary structure; the interven-

ing chain joining two hydrogen-bonded b-strands can

range from a tight turn to a long, structurally complex

stretch of polypeptide chain. Further, approximately

half the b-strands found in proteins are singletons and

do not form inter-strand hydrogen bonds with another

b-strand. Textbooks tend to blur this issue.

Typically, b-sheet is found in the interior of the

protein, although the outermost parts of edge-strands

usually reside at the protein’s water-accessible surface.

R EPETITIVE S ECONDARY S TRUCTURE :

THE P OLYPROLINE II H ELIX (P II )

When backbone dihedral angles are assigned repeating

f,c-values near (2708, þ 1408), the chain twists into

a left-handed helix with 3.0 residues per helical turn.

The name of this helix is derived from a poly-proline

homopolymer, in which the structure is forced by its

stereochemistry. However, a polypeptide chain can

adopt a P II helical conformation whether or not it

contains proline residues.

Unlike the better known a-helix, a P II helix has no

intrasegment hydrogen bonds, and it is not included in

the classical deﬁnition of secondary structure for this

reason. This extension of the deﬁnition is also needed in

the case of an isolated b-strand. Recent studies have

shown that the unfolded state of proteins is rich in

P II structure.

SECONDARY STRUCTURE IN PROTEIN ANALYSIS

N ONREPETITIVE S ECONDARY

S TRUCTURE: THE T URN

Turns are sites at which the polypeptide chain changes

its overall direction, and their frequent occurrence is the

reason why globular proteins are, in fact, globular.

Turns can be subdivided into b-turns, g-turns, and

tight turns. b-turns involve four consecutive residues,

with a hydrogen bond between the amide hydrogen of

the 4th residue and the carbonyl oxygen of the 1st

residue: N – H( i )···OyC( i 2 3). b-turns are further

subdivided into subtypes (e.g., Type I, I 0 , II, II 0 , III,…)

depending upon their detailed stereochemistry. g-turns

involve only three consecutive, hydrogen-bonded resi-

dues, N – H( i )· · ·OyC( i 2 2), which are further divided

into subtypes.

More gradual turns, known as “reverse turns” or

“tight turns,” are also abundant in protein structures.

Reverse turns lack intra-turn hydrogen bonds but

nonetheless, are involved in changes in overall chain

direction.

Turns are usually, but not invariably, found on the

water-accessible surface of proteins. Together, b,g-and

reverse turns account for about one-third of the

structure in globular proteins, on an average.

accept a protein’s three-dimensional coordinates as

input and provide its secondary structure components

as output.

I NHERENT A MBIGUITY IN

S TRUCTURAL I DENTIFICATION

It should be realized that objective criteria for structural

identiﬁcation can provide a welcome self-consistency,

but there is no single “right” answer. For example, turns

have been deﬁned in the literature as chains sites at

which the distance between two a-carbon atoms,

separated in sequence by four residues, is not more

than 7 ˚ , provided the residues are not in an a-helix:

distance[Ca( i )–Ca( i þ 3)] # 7 ˚ and Ca( i )–Ca( i þ 3)

not a-helix. Indeed, turns identiﬁed using this deﬁnition

agree quite well with one’s visual intuition. However,

the 7 ˚ threshold is somewhat arbitrary. Had 7.1 ˚ been

used instead, additional, intuitively plausible turns

would have been found.

P ROGRAMS TO I DENTIFY S TRUCTURE

FROM C OORDINATES

Many workers have devised algorithms to parse the

three-dimensional structure into its secondary structure

components. Unavoidably, these procedures include

investigator-deﬁned thresholds. Two such programs are

mentioned here.

N ONREPETITIVE S ECONDARY

S TRUCTURE : THE V-L OOP

V-loops are sites at which the polypeptide loops back on

itself, with a morphology that resembles the Greek letter

“V” although often with considerable distortion. They

range in length from 6 – 16 residues, and, lacking any

speciﬁc pattern of backbone-hydrogen bonding, can

exhibit signiﬁcant structural heterogeneity.

Like turns, V-loops are typically found on the outside

of proteins. On an average, there are about four such

structures in a globular protein.

The D atabase of S econdary S tructure

Assignments in P roteins

This is the most widely used secondary structure

identiﬁcation method available today. Developed by

Kabsch and Sander, it is accessible on the internet, both

from the original authors and in numerous implemen-

tations from other investigators as well.

The d atabase of s econdary s tructure assignments in

p roteins (DSSP) identiﬁes an extensive set of secondary

structure categories, based on a combination of back-

bone dihedral angles and hydrogen bonds. In turn,

hydrogen bonds are identiﬁed based on geometric criteria

involving both the distance and orientation between a

donor– acceptor pair. The program has criteria for

recognizing a-helix, 3 10 -helix, p helix, b-sheet (both

parallel and anti-parallel), hydrogen-bonded turns and

reverse turns. (Note: the p-helix is rare and has been

omitted from the secondary structure categories.)

Identiﬁcation of Secondary

Structure from Coordinates

Typically, one becomes familiar with a given protein

structure by visualizing a model – usually a computer

model – that is generated from experimentally deter-

mined coordinates. Some secondary structure types are

well deﬁned on visual inspection, but others are not. For

example, the central residues of a well-formed helix are

visually unambiguous, but the helix termini are subject

to interpretation. In general, visual parsing of the

protein into its elements of secondary structure can be

a highly subjective enterprise. Objective criteria have

been developed to resolve such ambiguity. These criteria

have been implemented in computer programs that

Pro tein S econdary S tructure Assignments

In contrast to DSSP, protein secondary structure assign-

ments (PROSS) identiﬁcation is based solely on back-

bone dihedral angles, without resorting to hydrogen

SECONDARY STRUCTURE IN PROTEIN ANALYSIS

bonds. Developed by Srinivasan and Rose, it is

accessible on the internet .

PROSS identiﬁes only a-helix, b-strand, and turns,

using standard f,c deﬁnitions for these categories.

Because hydrogen bonds are not among the identiﬁ-

cation criteria, PROSS does not distinguish between

isolated b-strands and those in a b-sheet.

proteins had been solved, these data-dependent f -values

ﬂuctuated signiﬁcantly as new structures were added to

the database. At this point there are more than 22 000

structures in the Protein Data Bank ( www.rcsb.org ), and

the f -values have reached a plateau.

D ATABASE- I NDEPENDENT P REDICTIONS:

THE H YDROPHOBICITY P ROFILE

Hydrophobicity proﬁles have been used to predict the

location of turns in proteins. A hydrophobicity proﬁle is

a plot of the residue number versus residue hydropho-

bicity, averaged over a running window. The only

variables are the size of the window used for averaging

and the choice of hydrophobicity scale (of which there

are many). No empirical data from the database is

required. Peaks in the proﬁle correspond to local

maxima in hydrophobicity, and valleys to local minima.

Prediction is based on the idea that apolar sites along the

chain (i.e., peaks in the proﬁle) will be disposed

preferentially to the molecular interior, forming a

hydrophobic core, whereas polar sites (i.e., valleys in

the proﬁle) will be disposed to the exterior and

correspond to chain turns.

Prediction of Protein

Secondary Structure from

Amino Acid Sequence

Efforts to predict secondary structure from amino acid

sequence dates back to the 1960s to the works of Guzzo,

Prothero and, slightly later, Chou and Fasman. The

problem is complicated by the fact that protein secondary

structure is only marginally stable, at best. Proteins fold

cooperatively, with secondary and tertiary structure

emerging more or less concomitantly. Typical peptide

fragments excised from the host protein, and measured in

isolation, exhibit only a weak tendency to adopt their

native secondary structure conformation.

P REDICTIONS B ASED ON E MPIRICALLY

D ETERMINED P REFERENCES

Motivated by early work of Chou and Fasman, this

approach uses a database of known structures to discover

the empirical likelihood, f

N EURAL N ETWORKS

More recently, neural network approaches to second-

ary structure prediction have come to dominate the

ﬁeld. These approaches are based on pattern-recog-

nition methods developed in artiﬁcial intelligence.

When used in conjunction with the protein database,

these

of ﬁnding each of the twenty

amino acids in helix, sheet, turn, etc . These likelihoods

are equated to the residue’s normalized frequency of

occurrence in a given secondary structure type, obtained

by counting. Using alanine in helices as an example

;

are

the

most

successful

programs

available

today.

A neural network is a computer program that

associates an input (e.g., a residue sequence) with an

output (e.g., secondary structure prediction) through a

complex network of interconnected nodes. The path

taken from the input through the network to the output

depends upon past experience. Thus, the network is said

to be “trained” on a dataset.

The method is based on the observation that amino

acid substitutions follow a pattern within a family of

homologous proteins. Therefore, if the sequence of

interest has homologues within the database of known

structures, this information can be used to improve

predictive success, provided the homologues are recog-

nizable. In fact, a homologue can be recognized quite

successfully when the sequence of interest and a putative

homologue have an aligned sequence identity of 25%

or more.

Neural nets provide an information-rich approach

to secondary structure prediction that has become

increasingly successful as the protein databank has

grown.

fraction Ala in helix ¼ occurrences of Ala in helices

occurrences of Ala in database

This fraction is then normalized against the corre-

sponding fraction of helices in the database:

fraction Ala in helix

fraction helices in database

f helix

Ala ¼

¼ occurrences of Ala in helices

occurrences of Ala in database

number of residues in helices

number of residues in database

A normalized frequency of unity indicates no pre-

ference – i.e., the frequency of occurrence of the given

residue in that particular position is the same as its

frequency at large. Normalized frequencies greater

than/less than unity indicate selection for/against the

given residue in a particular position.

These residue likelihoods are then used in combination

to make a prediction. When only a small number of

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