FOURTH EDITION
`
`ae
`
`ST o0 as
`
`Zipursky
`
`Matsudaira
`
`Baltimore
`
`Darnell
`
`MOLECULAR
`CELL
`BIOLOGY
`
`IPR2019-00797
`
`Media Connected
`
`:
`
`meal
`
`KASHIV EXHIBIT 1055
`
`Page 1
`
`KASHIV EXHIBIT 1055
`IPR2019-00797
`
`

`

`FOURTH EDITION
`
` eeeeeeeeeeeeee
`
`MOLECULAR
`CELE
`BIOLOGY
`
`Weaceal ttl
`
`Arnold Berk
`
`S. Lawrence Zipursky
`
`Paul Matsudaira
`
`David Baltimore
`
`James Darnell
`
`
`
`0)
`
`7.
`
`:
`
`-
`oy ce
`— Me ec
`Ha ye
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`"a
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`Media Connected :
`a a
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`A>
`Mme W. H. FREEMAN AND COMPANY
`
`|
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`" Ory
`Hi Me
`
`ica
`
`mary
`my)
`Paul Matsudaira
`viii gy
`i.
`'
`BU
`ae
`beicoArtty TSet
`mC!|ee
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`CT att
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`Page 2
`
`

`

`
`
`anataneeene
`
`ACCTCEA
`
`pagummenianmpemeencnmmrsibtictlltaAACATAAL
`
`EXECUTIVE Epiror: Sara Tenney
`DEVELOPMENT Eprrors: Katherine Abr, Ruth Steyn, Kay Ueno
`Eprroriat Assistant: Jessica Olshen
`EXECUTIVE MARKETING MANAGER: John A. Britch
`Project Eprror: Katherine Ahr
`Text AND Cover DesiGNEeR: Victoria Tomaselli
`Pacr Makeup: Michael Mendelsohn, Design 2000, Inc.
`Cover ILLUSTRATION: Kenneth Eward
`ILLUSTRATION CoorpINaToR: John Smith, Network Graphics; Tamara Goldman,Bill Page
`ILLusTRATIONS: Network Graphics
`PuHoto RESEARCHER: Jennifer MacMillan
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`Mep1A DevELOPERS: Sumanas, Inc.
`Composition: York Graphics Services, Inc.
`Manuracturinc: Von Hoffman Press
`
`Library of Congress Cataloging-in-Publication Data
`
`Molecular cell biology / Harvey Lodish p_
`p.
`cm.
`Includes bibliographical references.
`ISBN 0-7167-3136-3
`1, Cytology.
`2. Molecular biology.
`QHS581.2.M655
`1999
`5§71.6-de21
`
`[et al.] - 4th ed.
`
`I. Lodish, Harvey F.
`
`99-30831CIP
`
`© 1986, 1990, 1995, 2000 by W. H. Freeman and Company.All rights reserved.
`Nopart 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
`
`W. H. Freeman and Company
`41 Madison Avenue, New York, New York 10010
`Houndsmills, Basingstoke RG21 6X5, England
`
`Secondprinting, 2000
`
`Page 3 er
`
`
`
`
`
`Page 3
`
`

`

`
`
`Protein Structure
`and Function
`
`
`
`roteins, 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
`enzymescapable of catalyzing an incredible range of
`intracellular and extracellular chemical reactions, with
`a speed andspecificity 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 Saccharomyces cerevisiae, a simple
`unicellular eukaryote. The yeast genomeis predicted to
`encode about 6225 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 ofcell 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. Thefirst section
`examinesprotein architecture: the structure and chemistry of amino acids,
`A two-dimensional array of
`a-actinin molecules. the linkage of amino acids to formalinear chain, and the forces that guide
`
`folding of the chain into higher orders of structure. In the next section, we
`learn aboutspecial proteins that aid in the folding of proteins, modifications
`that occur after the protein chain is synthesized, and mechanisms that
`degradeproteins. 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.
`
`| | |
`
`|
`
`secaceaenneeeeeinaaenecinnneeeeaattnoaaeeaee
`;
`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 CONNECTIONS
`
`\C
`
`Focus: Chaperone-Mediated Folding
`Overview: Life Cycle of a Protein
`Technique: SDS Gel Electrophoresis
`Technique: Immunoblotting
`lassic Experiment 3.1: Bringing an Enzyme
`Back to Life
`
`Page 4
`
`

`

`
`
`These functionally diverse proteins playcritical roles
`in transfer of molecules and information across thelipid
`bilayer and in cell-cell interactions; their structures and
`functions will be discussed in greater detail in later chapters.
`We finish the chapter by describing the most commonly
`used techniquesin the biologist’s tool kit for isolating
`proteins and characterizing their properties. Our under-
`standing of biology critically depends on how wecan 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 complex moleculeslike 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 functionsefficiently 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 non-
`covalent interactions between regions in the linear sequence
`of amino acids. Only whena protein is in its correct three-
`dimensional 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-dimensional 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 @ carbon atom (C,) of amino acids, which is adjacent
`to the carboxyl group, is bonded to four different chemical
`groups: an amino (NH3) 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 aminoacids
`have this same general structure, but their side-chain groups
`vary in size, shape, charge, hydrophobicity, and reactivity.
`_ The aminoacidscan be considered the alphabet in which
`linear proteins are “written.” Students of biology must be
`familiar with the special properties of eachletter ofthis al-
`
`Hierarchical Structure of Proteins i 51
`
`MONOMER
`
`POLYMER
`
`
`
`Aminoacid
`
`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 four
`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 aminoacids, has 20*, or 160,000, possible
`sequences.
`
`phabet, which are determinedby the side chain. Amino acids
`can beclassified 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 groupstend to be on the surface of proteins; by
`interacting with water, they make proteins soluble in aque-
`ous solutions. In contrast, amino acids with nonpolarside
`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, arginine 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 ofa protein. A fifth aminoacid, histidine, has an im-
`idazole side chain, which has a pK, of 6.8, the pH of the
`cytoplasm.Asa result, smallshifts of cellular pH will change
`the charge of histidine side chains:
`
`CH,
`| n-H
`Co \
`ly
`C—H
`H~ Ny
`pH 5.8
`
`CH,
`| N-H
`Cc \
`a,
`C—H
`N
`pH 7.8
`
`H~
`
`The activities of many proteins are modulated by pH
`through protonationofhistidine side chains. Asparagine and
`glutamine are uncharged but have polar amide groups with
`extensive hydrogen-bondingcapacities. Similarly, serine and
`threonine are uncharged but have polar hydroxyl groups,
`which also participate in hydrogen bonds with other po-
`lar 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
`
`

`

`|
`52 | CHAPTER 3
`
`Protein Structure and Function
`
`HYDROPHILIC AMINO ACIDS
`
`Basic amino acids
`
`Polar amino acids with uncharged R groups
`
`COO-
`goo"
`“HAN—C—H
`“Tai-p-a
`CHa oe
`
`OH
`Serine
`(Ser or S)
`
`CH;
`Threonine
`(Thr or T)
`
`Gen
`coo”
`“HANC—H se
`.
`t
`Cc
`CH,
`fo
`|
`0
`Ccfi %&
`H,N No
`Glutamine
`(Gin or Q)
`
`Asparagine
`(Asn or N)
`
`H,N
`
`ee
`ae
`
`iC
`
`—NH
`
`‘cH4
`|
`c—n*
`H
`H
`
`coo”
`“hatl-o-8
`CHa
`GH
`
`goo"
`SHghE=A
`CHa
`CH
`
`sa
`
`o
`NH,”
`
`2
`Saath
`
`Lysine
`(Lys or K)
`
`NH,
`Arginine
`(Arg or R)
`
`Histidine
`(His or H)
`
`Acidic amino acids
`
`coo"
`Cc
`
`.:c
`
`oo™
`mic
`
`Goo"
`ae *H3N—
`
`7c
`
`oo™
`
`Aspartic
`
`Gluta
`
`
`
`HYDROPHOBIC AMINO ACIDS
`
`coo”
`Coo"
`a “HaN—C—H
`i
`i.
`x
`oH
`Ss
`
`H,C
`
`CH,
`
`goo"
`Goo"
`coo”
`“ee “Hee THAEH
`CH,
`CH
`GH
`C=CH
`NH
`
`|C
`
`H,
`
`OH
`
`ee
`coer
`goo"
`“Heit-H a *H3;N—C—H
`H,
`fs
`H
`¢ CH,
`CH
`
`;
`
`3
`
`Hg
`
`ea
`
`ess
`
`CH,
`
`Alanine
`(Ala or A)
`
`Valine
`(Val or V)
`
`Isoleucine
`(Ile or I)
`
`Leucine
`(Leu or L)
`
`Methionine
`(Met or M)
`
`Phenylalanine
`(Phe or F)
`
`Tyrosine
`(Tyr or Y)
`
`Tryptophan
`(Trp or W)
`
`A FIGURE3-2 The structures of the 20 common amino acids
`groupedinto three categories: hydrophilic, hydrophobic, and
`special amino acids. The side chain determines the characteris-
`tic properties of each aminoacid. Shownare the zwitterion
`forms, which exist at the pH of the cytosol. In parentheses are
`the three-letter and one-letter abbreviations for each amino acid.
`
`SPECIAL AMINO ACIDS
`
`Goo:
`*H,N—C—H
`7
`oO
`.
`SH
`Cysteine
`(Cys or C)
`
`oeH
`eo"
`C
` *H,N—C—H
`= H,N~ CH,
`i:
`Wo-—6
`a—— CH
`2
`
`2
`
`Glycine
`(Gly or G)
`
`Proline
`(Pro or P)
`
`Page 6—---—--———
`
`Page 6
`
`

`

`Hierarchical Structure of Proteins|53
`
`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, leucine, isoleucine, and me-
`thionine consist entirely of hydrocarbons, exceptforthe sul-
`fur atom in methionine,and all are nonpolar. Phenylalanine,
`tyrosine, and tryptophan have large bulky aromatic side
`groups. As explained in Chapter 2, hydrophobic molecules
`avoid water bycoalescing 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 environmentofthe 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—) to a second cysteine:
`
`|
`|
`a
`1
`B-CCH SH + HSCs—H
`rm
`re
`
`|
`|
`H—N
`N—H
`|
`|
`ies
`One
`7
`
`Regions within a protein chain or in separate chains some-
`times are cross-linked covalently through 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 asits
`R group. Its small size allows it to fit
`into tight spaces.
`Unlike any of the other common aminoacids, proline has
`a cyclic ring that is produced by formation of a covalent
`bond between its R group and the amino group on C,.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 known and predicted proteins encoded by the
`yeast genome have an average molecular weight (MW) of
`52,728 and contain, on average, 466 aminoacid residues.
`Assuming that these average values represent a “typical” eu-
`karyotic protein,
`then the average molecular weight of
`amino acids is 113, taking their average relative abundance
`i proteins into account. This is a useful number to re-
`
`member, as we can useit to estimate the numberof residues
`from the molecular weight of a protein or vice versa. Some
`amino acids are more abundantin proteins than other amino
`acids. Cysteine, tryptophan, and methionine are rare amino
`acids; together they constitute approximately 5 percent of
`the amino acids in a protein. Four amino acids—leucine,
`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 thelipid bilayer are enriched in hydrophobic aminoacids.
`
`Peptide Bonds Connect Amino Acids
`into Linear Chains
`
`linkage, the peptide
`Nature has evolved a single chemical
`bond, to connect aminoacids 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, C,,
`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 C, atoms. This leaves
`at opposite ends of the chain a free (unlinked) amino
`group (the N-terminus) and a free carboxyl group (the
`
`1
`it
`*H,N—C—C—O7 + “HNCeer
`Ry
`Re
`i
`
`H O
`H O
`|
`| it
`THN“GeGelbSO
`R, | R,
`
`Peptide
`bond
`
`(b)
`
`ao PEG ET TG
`“HANG-CKNGG Ga NoaiN c—C—oO-
`R,
`H
`> O
`3
`H
`4 2
`Rs;
`Amino end
`Carboxyl end
`(N-terminus)
`(C-terminus)
`
`A FIGURE 3-3 The peptide bond. (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 (R) extend from the backbone of
`a protein chain, in which the amino N, @ carbon, carbonyl
`carbon sequence is repeated throughout.
`
`
`
`Page 7
`
`

`

`bilizing forces hold the a helices, B strands, turns, and ran-
`domcoils in a compactinternal scaffold. Thus, a protein’s
`size and shape is dependent not only on its sequence but .
`also on the number, size, and arrangementofits 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)
`andrelative 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 ofcellular 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 othercell biopolymers
`like lipids, carbohydrates, and nucleic acids to form com-
`plex cell organelles.
`
`| |
`
`Protein Structure and Function
`54 | CHAPTER 3
`C-terminus). A protein chain is conventionally depicted with
`its N-terminal amino acid ontheleft and its C-terminal amino
`acid on the right (Figure 3-3b).
`Many terms are used to denote the chains formed by
`polymerization of aminoacids. 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 ofa cell.
`The size of a protein or a polypeptideis 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 commonlyis described in terms of
`fourhierarchical levels of organization. Theselevels areil-
`Graphic Representations of Proteins
`lustrated in Figure 3-4, which depicts the structure of hemag-
`Highlight Different Features
`glutinin, a surface protein on the influenza virus. This pro-
`Different ways of depicting proteins convey different types of
`tein binds to the surface of animalcells, including human
`information. The simplest way to represent three-dimensional
`cells, and is responsible for the infectivity of the flu virus.
`structure is to trace the course of the backbone atoms with
`The primary structure of a protein is the linear arrange-
`a solid line (Figure 3-Sa); the most complex model shows
`ment, or sequence, of amino acid residues that constitute the
`the location of every atom (Figure 3-5b; see also Figure 2-1a).
`polypeptide chain.
`The former shows the overall organization of the polypep-
`Secondarystructure refers to the localized organization
`tide chain without consideration of the amino acid side
`of parts of a polypeptide chain, which can assumeseveral
`chains; the latter details the interactions among atoms that
`different spatial arrangements. A single polypeptide may ex-
`form the backbone andthat stabilize the protein’s confor-
`hibit all types of secondary structure. Without any stabiliz-
`mation. Even though both viewsare useful, the elements of
`ing interactions, a polypeptide assumes a random-coil struc-
`secondary structure are not easily discerned in them.
`ture. However, when stabilizing hydrogen bonds form
`Another type of representation uses common shorthand
`between certain residues,
`the backbone folds periodically
`symbols for depicting secondary structure, cylinders for a
`into one of two geometric arrangements: an @ helix, which
`helices, arrows for 6 strands, andaflexible stringlike form
`is a spiral, rodlike structure, or a B sheet, a planar structure
`for parts of the backbone without any regularstructure (Fig-
`composed ofalignments of two or more B strands, which
`ure 3-5c). This type of representation emphasizes the orga-
`are relatively short, fully extended segments of the back-
`nization of the secondary structure of a protein, and vari-
`bone. Finally, U-shaped four-residue segments stabilized by
`ous combinations of secondary structures are easily seen.
`hydrogen bonds between their arms are called turns. They
`However, noneof these three ways of representing pro-
`are located at
`the surfaces of proteins and redirect the
`tein structure conveys much information about the protein
`polypeptide chain towardtheinterior. (These structures will
`surface, whichis of interest becausethis is where other mol-
`be discussed in greater detail later.)
`ecules bind to a protein. Computer analysis in which a wa-
`Tertiary structure,
`the next-higher level of structure,
`ter molecule is rolled around the surface of a protein can
`refers to the overall conformation of a polypeptide chain,
`identify the atomsthat are in contact with the watery envi-
`thatis, the three-dimensional arrangementofall the amino
`ronment. On this water-accessible surface, regions having a
`acids residues. In contrast to secondary structure, which is
`common chemical (hydrophobicity or hydrophilicity) and
`stabilized by hydrogen bonds,tertiary structureis stabilized
`electrical (basic or acidic) character can be mapped. Such
`by hydrophobic interactions between the nonpolar side
`models show the texture of the protein surface and the
`chains and, in some proteins, by disulfide bonds. These sta-
`
`Page 8——--
`
`Page 8
`
`

`

`Hierarchical Structure of Proteins|55
`
`68
`DALLGDPHCDVFONETWDLFVERSKAFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGV
`gf
`
`
`
`
`
`(a)
`
`yy
`
`195
`TONGGSNACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGIHHPSTNOEQTSL
`
`
`
`
`‘eatdseki add om y
`
`(b)
`
`DISTAL
`
`
`
`NH,
`
`
`PROXIMAL
`
`Globular
`domain
`
`Fibrous
`domain
`
`(c)
`
`Receptor
`site
`
`@
`
`domain correspond to the sequence shownin 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
`HAs (dark blue) with @ strands in HA,. Short turns and longer
`loops, which usually lie at the surface of the molecule, connect
`the helices and strands in a given chain. (c) 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 coiled-coil
`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.
`
`
`
`
`
`
`Viral
`membrane
`
`COOH
`
`A FIGURE 3-4 Four levels of structure in hemagglutinin,
`which is a long multimeric molecule whose three identical
`subunits are each composed of two chains, HA, and HAg.
`(a) Primary structureisillustrated by the amino acid sequence of
`residues 68-195 of HA. This region is used by influenza virus to
`bind to animalcells. 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 @ helices(light blue cylinders), 6 strands(light green
`arrows), and random coils (white strands). (b) 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
`Into two domains. The membrane-distal domain is folded into a
`Globular conformation. The blue and green segments in this
`
`
`
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`

`

`
`
`4
`
`56 | CHAPTER3
`
`Protein Structure and Function
`(e)
`
`(b)
`
`(d)
`
`A FIGURE 3-5 Various graphic representations of the struc-
`ture of Ras, a guanine nucleotide—-binding protein. Guanosine
`diphosphate, the substrate that is bound, is shownas a blue
`space-filling figure in parts (a)—(d). (a) The C,trace of Ras, which
`highlights the course of the backbone. Evident from this view Is
`how the polypeptide is packed into the smallest possible volume.
`(b) Ball-and-stick model of Ras showing the location of all atoms.
`(c) A schematic diagram of Ras showing how B strands (arrows)
`and «@ helices (cylinders) are organized in the protein. Note the
`
`turns and loops connectingpairs of helices and strands. (d) The
`water-accessible surface of Ras. Painted on the surface are
`regions of positive charge (blue) and negative charge (red). Here
`we see that the surface of a protein is not smooth but has lumps,
`bumps, and crevices. The molecular basis for specific binding
`interactionslies in the uneven distribution of charge over the
`surface of the protein. [Adapted from L. Tong et al., 1991, J. Mol.
`Biol, 217:503; courtesy of S. Choe.]
`
`distribution of charge, both of which are important param-
`eters of binding sites (Figure 3-Sd). 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 6
`
`sheets; the remainder of the molecule is in random coils and
`turns. Thus, a helices and 6 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 a Helix Polypeptide segments can assume a regular
`spiral, or helical, conformation, called the @ helix. In this
`
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`

`

`57
`
`|||
`
`Hierarchical Structure of Proteins
`
`such helices the hydrophobic residues, although apparently
`randomly arranged, occurin a regular pattern (Figure 3-7).
`One way of visualizing this arrangement is to look down
`the center of an @ 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 residuesall lie on one side of the wheel and
`the hydrophilic ones on the otherside.
`Amphipathic a helices are importantstructural elements
`in fibrous proteins found in a watery environment. In a
`coiled-coil region of a protein, the hydrophobic surface of
`the a 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 f strand, ran-
`dom coil, or another @ helix. As we discuss later, amphi-
`pathic 6 strands line the walls of an ion channelin thecell
`membrane.
`
`The B Sheet Another regular secondary structure, the B
`sheet, consists of laterally packed 6 strands. Each ® strand
`is a short (5—8-residue), nearly fully extended polypeptide chain.
`Hydrogen bonding between backbone atoms in adjacent B
`
`
`
`A FIGURE 3-7 Regions of an @ 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. Malashkevich.]
`
`
`
`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 arrangementof 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 having 3.6
`amino acids per turn (Figure 3-6). The stable arrangement
`of amino acidsin the @ helix holds the backboneas a rod-
`like cylinder from whichthe 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 hy-
`drophobicity or hydrophilicity.
`In many @ 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 residues/turn
`
`4 FIGURE 3-6 Model of the a helix. The polypeptide backbone
`is folded into a spiral that is held in place by hydrogen bonds
`[black dots) between backbone oxygen atoms and hydrogen
`atoms. Note that all the hydrogen bonds have the same polarity.
`The outer surface of the helix is covered by the side-chain
`R groups.
`
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`58 | CHAPTER 3
`
`
`
`Protein Structure and Function Face view
`
`
`Sideview
`
`‘?
`
`(a) A simple two-stranded B sheet
`A FIGURE 3-8 6 sheets.
`with antiparallel B strands. A sheetis stabilized by hydrogen bonds
`(black dots) between the £ strands. The planarity of the peptide
`bond forces a f sheet to be pleated; hence, this structure is also
`called a B pleated sheet, or simply a pleated sheet. (b) Side view
`of a B sheet showing how the R groups protrude above and
`below the plane of the sheet. (c) Modelof binding site in class |
`MHC(major histocompatibility complex) molecules, which are
`involved in graft rejection. A sheet comprising eight antiparallel 6
`strands (green) forms the bottom of the binding cleft, which is
`lined by a pair of a helices (blue). A disulfide bond is shown as
`two connected yellow spheres. The MHCbindingcleft is large
`enough to bind a peptide 8-10 residues long.[Part (b) adapted from
`C. Branden and J. Tooze, 1991, Introduction to Protein Structure, Garland.]
`
`strands, within either the same or different polypeptide
`chains, forms a B sheet (Figure 3-8a). Like a helices, B
`strands havea polarity defined by the orientation of the pep-
`tide bond. Therefore, in a pleated sheet, adjacent # 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, 6 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
`canslip over one another. However, they are also resistant
`to breakage because the peptide backboneis aligned paral-
`lel with the fiber axis.
`Turns Composed ofthree 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 bendthatredirects
`the polypeptide backbone back toward theinterior. 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
`backboneto fold into a tight U-shaped structure. Without
`turns, a protein would belarge, extended,andloosely 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
`Manyproteins contain one or more motifs built from par-
`ticular combinations of secondarystructures. 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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`

`

`4
`3
`
`¢4.‘
`
`(a)
`
`Hierarchical Structure of Proteins|59
`
`4 FIGURE 3-9 Secondary-structure
`motifs.
`(a) The coiled-coil motif (left)
`is characterized by two or more
`helices wound around one another.
`In some DNA-binding proteins, like
`c-Jun, a two-stranded coiled coil
`is
`responsible for dimerization(right).
`Each helix in a coiled coil has a
`repeated heptad sequence.
`LASTANMLREQVAQL
`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 which the hydro-
`phobic side chains are shownin red.
`(b) The helix-loop-helix motif occurs
`in many calcium-binding proteins.
`Oxygen-containing R groups of
`residues in the loop form a ring
`around a Ca** ion. The 14-aa loop
`sequence (right) is rich in invariant
`hydrophilic residues. (c) The zinc-
`finger motif is present in many
`proteins that bind nucleic acids. A
`Zn** ion is held betweena pair of B
`strands (green) an