FOURTH EDITION
`
`Lodish
`
`Berk
`
`MOLECULAR
`
`
`
`Zipursky
`
`Matsudaira
`
`BaHimore
`
`DarneH
`
`Media Connected .
`
`KASHIV EXHIBIT 1055
`
`|PR2019-00791
`
`Page 1
`
`KASHIV EXHIBIT 1055
`IPR2019-00791
`
`

`

`FOURTH EDITION
`
`
`
`MOLECULAR
`
`—l——-—-_——‘——-¢w——-_-
`
`CELL
`
`BIO LO GY
`
`
`
`Harvey Lodish
`
`Arnold Berk
`
`S. Lawrence Zipursky
`
`Paul Matsudaira
`
`David Baltimore
`
`James Darnell
`
`
`
`,
`.
`.
`lim
`P"
`I HI.“
`" n.
`.1.,,l,;'('.f,‘;,fj,
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`an)...“ llama-"“13
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`
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`
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`I
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`Media Connected :
`______._________
`g
`..-.* -
`I}: w. H. FREEMAN AND COMPANY’
`
`
`
`Page 2
`
`

`

`EXECUTIVE EDITOR: Sara Tenney
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`MANUFACTURING: Von Hoffman Press
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`Library of Congress Cataloging—in—I’ublicarion Data
`
`
`
`Molecular cell biologyI'Harvey Lodish p [et al.] — 4th ed.
`Pv
`011-
`_
`Includes bibliographical references.
`ISBN Ov7167v3136—3
`1. Cytology.
`2. Molecular biology.
`QH581.2.M655
`1999
`571.6—dc21
`
`I. Lodish, Harvey F.
`
`99-30831CIP
`
`© 1986, 1990, 1995, 2000 by W. H. Freeman and Company. All rights reserved.
`
`NO part Of this book may be reproduced by any mechanical, photographic, or
`electronic process, or in the form of a phonographic recording, nor may it be
`stored in a retrieval system, transmitted, or otherwise copied for public or private
`use, without written permission from the publisher.
`
`Printed in the United States of America
`
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`Houndsmills, Basingstoke RG21 6XS, England
`
`Second printing, 2000
`
`Page 3
`
`—__ -—_._.._———_l-_
`
`Page 3
`
`

`

`
`
`Protein Structure
`and Function
`
`
`
`Proteins, the working molecules of a cell, carry out the program of
`
`
`activities encoded by genes. This program requires the coordinated
`effort of many different types of proteins, which first evolved as
`rudimentary molecules that facilitated a limited number of chemical reactions.
`Gradually, many of these primitive proteins evolved into a wide array of
`enzymes capable of catalyzing an incredible range of
`intracellular and extracellular chemical reactions, with
`a speed and specificity that is nearly impossible to
`attain in a test tube. Other proteins acquired numerous
`structural, regulatory, and other functions. For a flavor
`of the various roles of proteins in today’s organisms, we
`can look to the yeast Saccharomyccs cercvisz'ae, a simple
`unicellular eukaryote. The yeast genome is predicted to
`encode about 622.5 proteins (see Table 7-3). On the
`basis of their sequences, 17 percent are estimated to be
`involved in metabolism, the synthesis or degradation of
`cell building blocks; 30 percent, in cellular organization
`and biogenesis of cell organelles and membranes; and
`10 percent, in transporting molecules across membranes.
`In this chapter, we will study how the structure of a
`protein gives rise to its function. The first section
`examines protein architecture: the structure and chemistry of amino acids,
`the linkage of amino acids to form a linear chain, and the forces that guide
`folding of the chain into higher orders of structure. In the next section, we
`learn about special proteins that aid in the folding of proteins, modifications
`that occur after the protein chain is synthesized, and mechanisms that
`degrade proteins. In the third section, we illustrate several key concepts in
`the functional design of proteins, using antibodies and enzymes as examples.
`A separate section is devoted to the general characteristics of membrane
`proteins, which reside in the lipid bilayer surrounding cells and organelles.
`
`A1wo~dimensional array of
`a-actinin molecules.
`
`OUTLINE
`
`3.1 Hierarchical Structure of Proteins 51
`3.2 Folding, Modification, and Degradation of
`Proteins 62
`
`3.3 Functional Design of Proteins 68
`
`3.4 Membrane Proteins 78
`3.5 Purifying, Detecting, and Characterizing
`Proteins 83
`
`MEDIA CDNNEETIDNS ffi
`Focus: Ehapernne-Mediated Folding
`
`Dverview: Life Cycle nf in Protein
`
`Technique: EDS Gel Electrophoresis
`
`Technique: lmrnunnhlnttlng
`'1
`Classic Experiment 3.]: Bringing an Enzyme
`Back to Life
`
`
`
`
`
`Page 4
`
`

`

`
`
`These functionaily diverse proteins play critical roles
`in transfer of molecules and information across the lipid
`bilayer and in cell—celi interactions; their structures and
`functions will be discussed in greater detail in later chapters.
`We finish the chapter by describing the most commonly
`used techniques in the biologist’s tool kit for isolating
`proteins and characterizing their properties. Our under—
`standing of biology critically depends on how we can ask
`a question and test it experimentally.
`
`3.1 Hierarchical Structure
`of Proteins
`
`Proteins are designed to bind every conceivable molecule—
`from simple ions to large compiex molecules like fats, sug—
`ars, nucleic acids, and other proteins. They catalyze an ex-
`traordinary range of chemical reactions, provide structural
`rigidity to the cell, control flow of material through mem—
`branes, regulate the concentrations of metabolites, act as
`sensors and switches, cause motion, and control gene func-
`tion. The three—dimensional structures of proteins have
`evolved to carry Out these functions efficiently and under
`precise control. The spatial organization of proteins, their
`shape in three dimensions, is a key to understanding how
`they work.
`One of the major areas of biological research today is
`how proteins, constructed from only 20 different amino
`acids, carry out the incredible array of diverse tasks that
`they do. Unlike the intricate branched structure of carbohy-
`drates, proteins are single, unbranched chains of amino acid
`monomers. The unique shape of proteins arises from none
`covalent interactions between regiOns in the linear sequence
`of amino acids. Only when a protein is in its correct three—
`dimensionai structure, or conformation, is it able to func-
`tion efficiently. A key concept in understanding how pro~
`teins work is that function is derived from three-dimensional
`structure, and three—dimensions:r structure is specified by
`amino acid sequence.
`
`The Amino Acids Composing Proteins
`Differ Only in Their Side Chains
`
`Amino acids are the monomeric building blocks of proteins.
`The 0: carbon atom ((30,) of amino acids, which is adjacent
`to the carboxyl group, is bonded to four different chemical
`grOups: an amino (NHZJ group, a carboxyl (COOH) group,
`a hydrogen (H) atom, and one variable group, called a side
`chain or R group (Figure 3-1). All 20 different amino acids
`have this same general structure, but their side—chain groups
`Vary in size, shape, charge, hydrophobicity, and reactivity.
`_ The amino acids can be considered the alphabet in which
`llnear proteins are “written.” Students of biology must be
`familiar with the special properties of each letter of this al—
`
`Hierarchical Structure of Proteins i 51
`
`MONOMER
`H0
`||
`
`POLYMER
`
` Amino acid
`
`A FIGURE 3-1 Amino acids. the monomeric units that link
`together to form proteins. have a common structure. The
`a carbon atom {green} of each amino acid is bonded to {our
`different chemical groups and thus is asymmetric. The side chain,
`or R group {red}, is unique to each amino acid. The diversity of
`natural proteins reflects different linear combinations of the 20
`naturally occurring amino acids. The short peptide shown here,
`containing only four amino acids, has 20”, or 160,000. possible
`sequences.
`
`phabet, which are determined by the side chain. Amino acids
`can be classified into a few distinct categories based primar-
`ily on their solubility in water, which is influenced by the
`polarity of their side chains (Figure 3-2}. Amino acids with
`polar side groups tend to be on the surface of proteins; by
`interacting with water, they make proteins soluble in aque—
`ous solutions. In contrast, amino acids with nonpolar side
`groups avoid water and aggregate to form the water—
`insoluble core of proteins. The polarity of amino acid side
`chains thus is one of the forces responsible for shaping the
`final three-dimensional structure of proteins.
`Hydrophilic, or water—soluble, amino acids have ionized
`or polar side chains. At neutral pH, ntgim'ne and lysine are
`positively charged; aspartic acid and glutamic acid are neg-
`atively charged and exist as aspartate and glutamate. These
`four amino acids are the prime contributors to the overall
`charge of a protein. A fifth amino acid, bistidiue, has an im-
`idazole side chain, which has a pK,l of 6.8, the pH of the
`cytoplasm. As a result, small shifts of cellular pH will change
`the charge of histidine side chains:
`
`CH2
`|
`Isl/H
`C/ \
`C—H
`i... r
`I-I/ NEH
`pH 5.8
`
`CH2
`l N/H
`C/ \
`C-—H
`('L /
`N
`pH 7.8
`
`H/
`
`The activities of many proteins are modulated by pH
`throrigh protonation of histidine side chains. Asparngiue and
`glutamine are uncharged but have polar amide groups with
`extensive hydrogen—bonding capacities. Similarly, serine and
`tbreonine are uncharged but have polar hydroxyl groups,
`which also participate in hydrogen bonds with other po-
`iar molecules. Because the charged and polar amino acids
`are hydrophilic, they are usually found at the surface of a
`water~soluble protein, where they not only contribute to
`
`Page 5
`
`Page 5
`
`

`

`
`
`l
`52 1 CHAPTER 3
`
`Plotein Structure and Function
`
`HYDROF‘HILIC AMINO ACIDS
`
`Basic amino acids
`
`Polar amino acids with uncharged R groups
`
`(1300"
`+H3N—ClZ—H
`(IIHQz
`(IJH2
`(EH2
`CH
`I
`24-
`NH3
`
`Lysine
`[Lye or Kl
`
`(I300‘
`+H3N—C12—H
`(IJH2
`(IZH2
`{EH2
`NH
`I
`$=NH2
`NH:
`Arginlne
`[Arg or B)
`
`+
`
`(IIOO’
`+H3N—{l3—H
`(13H2
`C—Nh
`llI—N%
`
`H
`
`H
`
`CH
`
`Histidine
`[His or H}
`
`Acidic amino acids
`
`C00_
`(I:
`+H N
`H
`e _ _
`|
`i”2
`C00"
`
`Aspartic
`
`C00“
`+H N (I:
`H
`3 _ _
`|
`‘iHZ
`('leCOO—
`Glutamic
`
`CIIZOO'
`+H3N—Cll-H
`(sz
`OH
`[Ser orS]
`
`Serine
`
`(IZOD—
`+H3N—(IZ—H
`H—Cli—OH
`CH3
`{Thr orT}
`
`Threonine
`
`(I300‘
`+H3N—rlz—H
`[EH2
`/C\\
`O
`
`“Z“
`
`Asparagine
`men or N]
`
`('300"
`+H3N—(Il—H
`(EH2
`(EH2
`C/ \\
`0
`
`HQN
`
`Glutamin'e
`{Gin or 0)
`
`('300‘
`+H3N—(IJ—H
`CH3
`
`(1200‘
`+H3N—CII—H
`fig
`
`HaC
`
`CH3
`
`HYDROF‘HOBIC AMINO ACIDS
`
`([300—
`+H3N—(II—H
`H_(l:—CH3
`(Isz
`CH3
`
`$00"
`+H3N—(i2-H
`(lin
`/cfi
`{3H3
`
`H30
`
`(1200’
`+HSN—tl:—H
`('le
`(EH:
`5|;
`
`CH3
`
`$00”
`+H3N—(II—H
`CH2
`
`CIOO‘
`+H3N—(l3—H
`CH2
`
`OH
`
`(IZOO‘
`+H3N—(13—-H
`(sz
`(3qu
`__ NH
`
`Alanine
`[Ala or A}
`
`Valine
`[Val or V}
`
`Isoleucine
`(lie or I]
`
`Leueine
`lLeu or L]
`
`Methionine
`{Met or M]
`
`Phenylalanine
`lPhe or Fl
`
`Tyrosine
`{Tvr or VII
`
`Twptophan
`iTrp or W}
`
`A FIGURE 3-2 The structures of the 20 common amino acids
`grouped into three categories: hydrophilic. hydrophobic, and
`special amino acids. The side chain determines the characteris—
`tic properties of each amino acid. Shown are the zwittericn
`forms, which exist at the pH of the cvtosoi. In parentheses are
`the three-letter and one—letter abbreviations for each amino acid.
`
`SPECIAL AMINO ACIDS
`
`COO‘
`+H3N~(13—nH
`|
`{EH2
`SH
`Cysteine
`(Cys or (:1
`
`COO"
`+H3N—(IZ—H
`|
`H
`
`Glycine
`[Gly or G)
`
`COO-
`(l3/H
`“‘“CHZ
`{13H
`2
`
`H1N/
`H g
`2
`
`Prelim
`[Pro or Pl
`
`1
`
`Page 6—___
`
`.
`
`.
`
`Page 6
`
`

`

`the solubility of the protein in water but also form binding
`sites for charged molecules.
`Hydrophobic amino acids have aliphatic side chains,
`which are insoluble or only slightly soluble in water. The
`side chains of alanine, valine, lencfne, isolencine, and me-
`thionine consist entirely of hydrocarbons, except for the sul—
`fur atom in methionine, and all are nonpolar. Phenylalanine,
`tyrosine, and tryprophan have large bulky aromatic side
`groups. As explained in Chapter 2, hydrophobic molecules
`avoid water by coalescing into an oily or waxy droplet. The
`same forces cause hydrophobic amino acids to pack in the
`interior of proteins, away from the aqueous environment.
`Later in this chapter, we will see in detail how hydrophobic
`residues line the surface of membrane proteins that reside
`in the hydrophobic environment of the lipid bilayer.
`Lastly, cysteine, glycine, and proline exhibit special roles
`in proteins because of the unique properties of their side
`chains. The side chain of cysteine contains a reactive
`sulfhydryl group (—SH), which can oxidize to form a disul-
`fide bond (—S—S—l to a second cysteine:
`
`|
`l
`p—H
`n—H
`H—p—Cnsz+Hs—Ggq—H
`i=0
`i=0
`
`l
`1
`n—H
`H—T
`H‘$—CHf—S—S—CHfF$—H
`o=c
`C=O
`
`Regions within a protein chain or in separate chains SOme-
`times are cross—linked covalently thrortgh disulfide bonds.
`Although disulfide bonds are rare in intracellular proteins,
`they are commonly found in extracellular proteins, where
`they help maintain the native, folded structure. The small—
`est amino acid, glycine, has a single hydrogen atOm as its
`R group. Its small size allows it to fit
`into tight spaces.
`Unlike any of the other common amino acids, proline has
`a cyclic ring that is produced by formation of a covalent
`bond between its R group and the amino group on Ca. Pro-
`line is very rigid, and its presence creates a fixed kink in a
`protein chain. Proline and glycine are sometimes found at
`points on a protein’s surface where the chain loops back into
`the protein.
`The 6225 lcnown and predicted proteins encoded by the
`East genome have an average molecular weight (MW) of
`52,728 and contain, On average, 466 amino acid residues.
`Assuming that these average values represent a “typical” eu~
`k61ryoric protein,
`then the average molecular weight of
`amino acids is 113, taking their average relative abundance
`”1 proteins into account. This is a useful number to re-
`
`Hicrarchical Structure of Proteins
`
`53
`
`member, as we can use it to estimate the number of residues
`from the molecular weight of a protein or vice versa. SOme
`amino acids are more abundant in proteins than other amino
`acids. Cysteine, tryptophan, and methionine are rare amino
`acids; together they constitute appr0ximately 5 percent of
`the amino acids in a protein. Four amino acids—lencine,
`serine,
`lysine, and glutamic acid—are the most abundant
`amino acids,
`totaling 32 percent of all
`the amino acid
`residues in a typical protein. However, the amino acid com—
`position of proteins can vary widely from these values. For
`example, as discussed in later sections, proteins that reside
`in the lipid bilayer are enriched in hydrophobic amino acids.
`
`Peptide Bonds Connect Amino Acids
`into Linear Chains
`
`linkage, the peptide
`Nature has evolved a single chemical
`bond, to connect amino acids into a linear, unbranched chain.
`The peptide bond is formed by a condensation reaction be—
`tween the amino group of one amino acid and the carboxyl
`group of another (Figure 3~3a). The repeated amide N, Ca,
`and carbonyl C atoms of each amino acid residue form the
`backbone of a protein molecule from which the various
`side-chain groups project. As a consequence of the peptide
`linkage,
`the backbone has polarity, since all
`the amino
`groups lie to the same side of the Cu atoms. This leaves
`at opposite ends of the chain a free (unlinked) amino
`group (the N-terminus) and a free carboxyl group (the
`
`(all
`
`H
`D
`H 0
`*H,N—-t|:,x—d—o— + +H,N—(|:a—(ll—0’
`l.
`l.
`
`iE‘fiHZO
`
`H 0
`H O
`+H,N—(':;il—N—tl:;tli—O‘
`l.
`ll l,
`
`Peptide
`bond
`
`lb}
`
`H 0
`H
`H
`H O
`H
`H
`+H3N—l5l—N—lrc—l—lrlw—lgc—l
`i. itl
`t iii
`Amino end
`lN-terminus)
`
`H 0
`('3;ii—o*
`t
`Carboxyl and
`(CI—terminus}
`
`A FIGURE 3-3 The peptide band. {a} A condensation
`reaction between two amino acids forms the peptide bond.
`which links all the adjacent residues in a protein chain.
`{b} Side-chain groups {Fl} extend from the backbone of
`a protein chain, in which the amino N, or carbon. carbonyl
`carbon sequence is repeated throughout.
`
`Page?
`
`Page 7
`
`

`

`bilizing forces hold the or helices, ,8 strands, turns, and ran~
`dom coils in a compact internal scaffold. Thus, a protein’s
`size and shape is dependent not only on its sequence but _
`also on the number, size, and arrangement of its secondary
`structures. For proteins that consist of a single polypeptide
`chain, monomeric proteins, tertiary structure is the highest
`level of organization.
`Multimeric proteins contain two or more polypeptide
`chains, or subunits, held together by noncovalent bonds.
`Quaternary structure describes the number (stoichiometry)
`and relative positions of the subunits in a multimeric pro—
`tein. Hemagglutinin is a trimer of three identical subunits;
`other multimeric proteins can be composed of any number
`of identical or different subunits.
`In a fashion similar to the hierarchy of structures that
`make up a protein, proteins themselves are part of a hier-
`archy of cellular structures. Proteins can associate into larger
`structures termed macromolecular assemblies. Examples of
`such macromolecular assemblies include the protein coat of
`a virus, a bundle of actin filaments, the nuclear pore com—
`plex, and other large submicroscopic objects. Macromolec-
`ular assemblies in turn combine with other cell biopolymers
`like lipids, carbohydrates, and nucleic acids to form com—
`plex cell organelles.
`
`Graphic Representations of Proteins
`Highlight Different Features
`Different ways of depicting proteins convey different types of
`information. The simplest way to represent three—dimensional
`structure is to trace the course of the backbone atoms with
`a solid line (Figure 3—5a); the most complex model shows
`the location of every atom (Figure 3-5b; see also Figure 2-13).
`The former shows the overall organization of the polypep—
`tide chain without consideration of the amino acid side
`chains, the latter details the interactions among atoms that
`form the backbone and that stabilize the protein’s confor—
`mation. Even though both views are useful, the elements of
`secondary structure are not easily discerned in them.
`Another type of representation uses common shorthand
`symbols for depicting secondary structure, cylinders for a:
`helices, arrows for ,8 strands, and a flexible stringlike form
`for parts of the backbone without any regular structure (Fig-
`ure 3~5c}. This type of representation emphasizes the orga—
`nization of the secondary structure of a protein, and vari-
`ous combinations of secondary structures are easily seen.
`However, none of these three ways of representing pro-
`tein structure conveys much information about the protein
`surface, which is of interest because this is where other mol—
`ecules bind to a protein. Computer analysis in which a wa-
`ter molecule is rolled around the surface of a protein can
`identify the atoms that are in contact with the watery envi-
`ronment. On this water—accessible surface, regions having a
`common chemical (hydrophobicity or hydrophilicity} and
`electrical (basic or acidic} character can be mapped. Such
`models show the texture of the protein surface and the
`
`l lI
`
`54 l CHAPTER 3
`
`Protein Structure and Function
`
`C-terminus}. A protein chain is conventionally depicted with
`its N-terminal amino acid 0n the left and its C~terminal amino
`acid on the right (Figure 3—3b}.
`Many terms are used to denote the chains formed by
`polymerization of amino acids. A short chain of amino acids
`linked by peptide bonds and having a defined sequence
`is a peptide;
`longer peptides are referred to as polypep-
`tides. Peptides generally contain fewer than 20—30 amino
`acid residues, whereas polypeptides contain as many as
`4000 residues. We reserve the term protein for a poly-
`peptide (or a complex of polypeptides) that has a three
`dimensional structure. It is implied that proteins and pep—
`tides represent natural products of a cell.
`The size of a protein or a polypeptide is reported as its
`mass in daltons (a dalton is 1 atomic mass unit) or as its mo-
`lecular weight (a dimensionless number}. For example, a
`10,000-MW protein has a mass of 10,000 daltons (Da), or
`10 kilodaltons (kDa). In the last section of this chapter, we
`will discuss different methods for measuring the sizes and
`other physical characteristics of proteins.
`
`Four Levels of Structure Determine
`the Shape of Proteins
`The structure of proteins commonly is described in terms of
`four hierarchical levels of organization. These levels are il~
`lustrated in Figure 3—4, which depicts the structure of hemag—
`glutinin, a surface protein on the influenza virus. This pro-
`tein binds to the surface of animal cells, including human
`cells, and is responsible for the infectivity of the flu virus.
`The primary structure of a protein is the linear arrange
`ment, or sequence, of amino acid residues that constitute the
`polypeptide chain.
`Secondary structure refers to the localized organization
`of parts of a polypeptide chain, which can assume several
`different spatial arrangements. A single polypeptide may ex-
`hibit all types of secondary structure. Without any stabiliz-
`ing interactions, a polypeptide assumes a random-coil struc—
`ture. However, when stabilizing hydrogen bonds form
`between certain residues,
`the backbone folds periodically
`into one of two geometric arrangements: an a: helix, which
`is a spiral, rodlike structure, or a ,8 sheet, a planar structure
`composed of alignments of two or more ,8 strands, which
`are relatively short, fully extended segments of the back-
`bone. Finally, U—shaped four-residue segments stabilized by
`hydrogen bonds between their arms are called turns. They
`are located at
`the surfaces of proteins and redirect the
`polypeptide chain toward the interior. (These structures will
`be discussed in greater detail later.)
`Tertiary structure,
`the next-higher level of structure,
`refers to the overall conformation of a polypeptide chain,
`that is, the three~dimensional arrangement of all the amino
`acids residues. in contrast to secondary structure, which is
`sta bilizcd by hydrogen bonds, tertiary structure is stabilized
`by hydrophobic interactions between the nonpolar side
`chains and, in some proteins, by disulfide bonds. These sta-
`
`Page 8
`
`
`
`Page 8
`
`

`

`Hierarchical Structure of Proteins
`
`55
`
`lai
`
`es
`DAL LGDPHCDVFON ETWDLFVE RS KAFSNCYPYDV PDYAS LRS LVAS SGTLEFITEGFTWTGV
`..
`
`
`
`
`mar-w
`
`*7"
`
`195
`TONGGSNACKHGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNOEOTSL
`
`
`
`‘I'II'il . IIII iii
`
`
`
`Receptor
`site
`
`.
`
`{c}
`
`lbi
`
`
`
`Globular
`domain
`
`Fibrous
`domain
`
`PROXIMAL
`
`
`
`Viral
`
`membrane
`
`COOH
`
`A FIGURE 3—4 Four levels of structure in hemegglutinin.
`which is a long multimeric molecule whose three identical
`sIJbunite are each composed of two chains. HA1 and HA2.
`lal Primary structure is illustrated by the amino acid sequence of
`residues 68—195 of HA1. This region is used by influenza virus to
`bind to animal cells. The one—letter amino acid code is used.
`Secondary structure is represented diagrammatically beneath the
`Sequence, showing regions of the polypeptide chain that are
`folded into 0: helices {light blue cylinders}. ,8 strands (lighi green
`ariOWSi, and random coils (white strands}. (bi Tertiary structure
`Constitutes the folding of the helices and strands in each HA
`SUbunit into a compact structure that is 13.5 nm long and divided
`WED two domains. The membrane-distal domain is folded into a
`QIObUIar conformation. The blue and green segments in this
`
`domain correspond to the sequence shown in part {a}. The proxi-
`mal domain, which lies adjacent to the viral membrane, has a
`stemlike conformation due to alignment of two long helices of
`HA2 {dark blue} with B strands in HA1. Short turns and ionger
`loops, which usually lie at the surface of the molecule, connect
`the helices and strands in a given chain. lci The quaternary
`structure comprises the three subunits of HA; the structure is
`stabilized by lateral interactions among the long helices {dark
`blue} in the subunit stems, forming a triple-stranded coiledvcoii
`stalk. Each of the distal globular domains in trimeric hemagglutinin
`has a site {red} for binding sialic acid molecules on the surface
`of target cells. Like many membrane proteins. HA has several
`covalently bound carbohydrate {CHO} chains.
`
`
`
`Page 9
`
`

`

`
`
`4
`
`56 i CHAPTER 3
`
`Protein Structure and Function
`
`lb}
`
`
`
`
`{cl}
`
`A FlGURE 3-5 Various graphic representations of the struc-
`ture of Has, a guanine nucieotida—binding protein. Guanosine
`diphosphate, the substrate that is bound, is shown as a blue
`space-filling figure in parts tal—ldl. {a} The (3,, trace of Rats, which
`highlights the course of the backbone. Evident from this view is
`how the polypeptide is packed into the smallest possible volume.
`{bl Ball-and-stick model of Flas showing the location of all atoms.
`to} A schematic diagram of Res showing how ,8 strands {arrowsl
`and a helices lcylindersl are organized in the protein. Note the
`
`turns and loops connecting pairs of helices and strands. ld‘l The
`water—accessible surface of Has. Painted on the surface are
`regions of positive charge {blue} and negative charge lredl. Here
`we see that the surface of a protein is not smooth but has lumps,
`bumps, and crevices. The molecular basis for specific binding
`interactions lies in the uneven distribution of charge over the
`surface of the protein. [Adapted from L. Tong et at, 1991. J. Mol.
`Biol. 21?:503; courtesy of S. Choe.l
`
`distribution of charge, both of which are important param—
`eters of binding sites [Figure 3—5d). This view represents
`a protein as seen by another molecule.
`
`Secondary Structures Are Crucial Elements
`of Protein Architecture
`In an average protein, 60 percent of the polypeptide chain
`exists as two regular secondary structures, a helices and B
`
`sheets; the remainder of the molecule is in random coils and
`turns. Thus, at helices and ,8 sheets are the major internal
`supportive elements in proteins. In this section, we explore
`the forces that favor formation of secondary structures. In
`later sections, we examine how these structures can pack
`into larger arrays.
`The or Helix Polypeptide segments can assmne a regular
`spiral, or helical, conformation, called the or helix. In this
`
`Page 10
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`Page 10
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`

`

`i |1
`
`57
`
`Hierarchical Structure of Proteins
`
`such helices the hydrophobic residues, although apparently
`randomly arranged, occur in a regular pattern (Figure 3—7).
`One way of visualizing this arrangement is to look down
`the center of an a helix and then project the amino acid
`residues onto the plane of the paper. The residues will ap—
`pear as a wheel, and in the case of an amphipathic helix,
`the hydrophobic residues all lie on one side of the wheel and
`the hydrophilic ones on the other side.
`Amphipathic or helices are important structural elements
`in fibrous proteins found in a watery environment. In a
`coiled~coil region of a protein, the hydrophobic surface of.
`the or helix faces inward to form the hydrophobic core, and
`the hydrophilic surfaces face outward toward the sur—
`rounding fluid. This same orientation of surfaces is also
`found in most globular proteins. A crucial difference is that
`the hydrophobic interaction could be with a ,8 strand, ran-
`dom coil, or another tr helix. As we discuss later, amphi—
`pathic ,8 strands line the walls of an ion channel in the cell
`membrane.
`
`The ,6 Sheet Another regular secondary structure, the 6
`sheet, consists of laterally packed B strands. Each 8 strand
`is a short (5—8—residue}, nearly fully extended polypeptide chain.
`Hydrogen bonding between backbone atoms in adjacent ,8
`
`
`
`A FIGURE 3-7 Regions of an a helix may be amphipathic.
`The five chains of cartilage oligomeric matrix protein associate
`into a coiled-coil fibrous domain through amphipathic a helices.
`Seen in cross section through a part of the domain, the hy-
`drophobic residues {gray} face the interior, and the hydrophilic
`residues {yellow} line the surface. This arrangement of hydropho—
`bic and hydrophilic residues is typical of proteins in an aqueous
`environment. [Courtesy of V. Malashkevichl
`
`secondary structure, the carbonyl oxygen of each peptide
`bond is hydrogen—bonded to the amide hydrogen of the
`amino acid four residues toward the C—terminus. This uni-
`form arrangement of bonds confers a polarity on a helix be—
`cause all the hydrogen-bond donors have the same orienta-
`tion. The peptide backbone twists into a helix haying 3.6
`amino acids per turn (Figure 3~6). The stable arrangement
`of amino acids in the a helix holds the backbone as a rod-
`like cylinder from which the side chains point outward. The
`hydrophobic or hydrophilic quality of the helix is deter—
`mined entirely by the side chains, because the polar groups
`of the peptide backbone are already involved in hydrogen
`bonding in the helix and thus are unable to affect its by—
`drophobicity or hydrophilicity.
`In many a helices hydrophilic side chains extend from
`one side of the helix and hydrophobic side chains from the
`opposite side, making the overall structure amphipathic. In
`
`
`
`
`3.6 residuesiturn
`
`A FIGURE 3-6 Model of the a helix. The polypeptide backbone
`is folded into a spiral that is held in place by hydrogen bonds
`ibIaCk dots} between backbone oxygen atoms and hydrogen
`atUrns. Note that all the hydrogen bonds have the same polarity.
`The outer surface of the helix is covered by the side-chain
`R groups,
`
`
`
`Page 11
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`i E
`
`58! CHAPTER 3
`
`Protein Structure and Function
`
`
`
`to)
`
` Face View
`
`
`Side view J
`
`{a} A simple two-stranded ,8 sheet
`A FIGURE 3-8 B sheets.
`with antiparallel ,8 strands. A sheet is stabilized by hydrogen bonds
`(black dots} between the ,B strands. The planarity of the peptide
`bond forces a ,6 sheet to be pleated; hence, this structure is also
`called a B pleated sheet, or simply a pieated sheet. (bi Side View
`of a ,8 sheet showing how the R groups protrude above and
`below the plane of the sheet. {cl Model of binding site in class i
`MHC {major histocompatibility complex} molecules, which are
`involved in graft rejection, A sheet comprising eight antiparallel ,3
`strands [green] forms the bottom of the binding cleft. which is
`lined by a pair of or helices {blue}. A disulfide bond is shown as
`two connected yellow spheres. The MHC binding cleft is large
`enough to bind a peptide 8—10 residues long. [Part {bl adapted from
`C. Branden and J. Tooze, 1991. introduction to Protein Structure, Garlandl
`
`strands, within either the same or different poiypeptide
`chains, forms a ,8 sheet {Figure 3~8a). Like or helices,
`,8
`strands have a polarity defined by the orientation of the pep—
`tide bond. Therefore, in a pleated sheet, adjacent ,8 strands
`can be oriented antiparallel or parallel with respect to each
`other. In both arrangements of the backbone, the side chains
`project from both faces of the sheet (Figure 3-8b).
`In some proteins, ,8 sheets form the floor of a binding
`pocket (Figure 3—8c). In many structural proteins, multiple
`layers of pleated sheets provide toughness. Silk fibers, for
`example, consist almost entirely of stacks of antiparallel ,6
`sheets. The fibers are flexible because the stacks of ,8 sheets
`can slip over one another. However, they are also resistant
`to breakage because the peptide backbone is aligned paral-
`lel with the fiber axis.
`
`Turns Composed of three or four residues, turns are com—
`pact, U-shaped secondary structures stabilized by a hydro-
`gen bond between their end residues. They are located on
`
`the surface of a protein, forming a sharp bend that redirects
`the polypeptide backbone back toward the interior. Glycine
`and proline are commonly present in turns. The lack of a
`large side chain in the case of glycine and the presence of
`a built-in bend in the case of proline allow the polypeptide
`backbone to fold into a tight U-shaped structure. Without
`turns, a protein would be large, extended, and loosely packed.
`A polypeptide backbone also may contain long bends, or
`loops. In contrast to turns, which exhibit a few defined struc-
`tures, loops can be formed in many different ways.
`
`Motifs Are Regular Combinations
`of Secondary Structures
`Many proteins contain one or more motifs built from par-
`ticular combinations of secondary structures. A motif is de-
`fined by a specific combination of secondary structures that
`has a particular topology and is organized into a charac-
`
`'Page 12
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`

`Hierarchical Structure of Proteins
`
`59
`
`4 FIGURE 3-9 Secondary-structure
`motifs.
`la} The coiled—coil motif (left)
`is characterized by two or more
`helices wound around one another.
`
`In some DNA—binding proteins, |i|<e
`c—Jun. a two—stranded coiled coil
`is
`responsible for dimerization {right}.
`Each helix in a coiled coil has a
`repeated heptad sequence.
`LASTANMLHEOVAOL
`1
`4
`1
`4
`1
`with a leucine or other hydrophobic
`residue (red) at positions 1 and 4,
`forming a hydrophobic stripe along
`the helix surface. The helices pair by
`binding along their hydrophobic
`stripes, as seen in both models
`displayed here, in wh