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Intro to Protein Structure, p. 1
BCMP201, 2001
Overview of Protein Structure
General References
Stryer, L. 1995. Protein Structure and Function. Chapter 2 in Biochemistry, fourth ed. (W.H. Freeman, New York).
Branden, C. and Tooze, J. 1991. Chapters 1 and 2, pp. 1-31 in Introduction to Protein Structure. (Garland Publishing, New
York).
Creighton T.E. 1994. Physical Interactions that Determine the Properties of Proteins. Chapter 4, pp. 139-165 in Proteins, 2nd
edition. (W.H. Freeman, New York).
Creighton T.E. 1994. Conformational Properties of Polypeptide Chains. Chapter 5, . pp. 171-1196 in Proteins, 2nd edition.
(W.H. Freeman, New York).
Creighton T.E. 1994. Proteins in Solution and in Membranes. Chapter 7, pp. 261-325 in Proteins, 2nd edition. (W.H. Freeman,
New York).
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The three-dimensional structures of small molecules are reasonably well defined by their covalent
bonding arrangement. Proteins and other macromolecules have markedly greater conformational
freedom by virtue of their large size and chemical complexity. Rotation about single bonds in a
polypeptide chain generates a vast number of potential alternative conformations. Proteins are unusual
among chemical polymers in that they adopt one folded conformation in solution. Steric repulsion
between neighboring amino acids partially restricts the number of conformations accessible to a peptide
chain. Weak noncovalent interactions among neighboring residues in the folded state determine the
native conformation. However, not all protein segments are stably folded under physiological
conditions; flexion or movement of certain regions may play a role in protein-protein interactions, the
generation of force, or in serving as an on-and-off switch for a protein’s function.
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We will first examine the chemical properties of polypeptides, emphasizing those features contributing to
their folded structure. The chemical parameters dictating native structure are complex and poorly
understood. Further insight into the “protein folding problem” can be gained by studying the diversity of
amino acid sequences present in families of structurally homologous proteins, in order to identify the
absolute sequence requirements for a given peptide fold. These efforts have led to improved methods
of predicting the native structures of peptides and proteins.
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Amino Acids and the Peptide Bond:
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The peptide bond is a key determinant of protein structure- Polypeptides are formed by a
ribosome-catalyzed condensation of amino acids, creating a linear chain with restricted flexibility. The
main chain atoms N, Cα, C and O, are common to all amino acids and these form the 'backbone', or
main chain, of the peptide. Peptide bonds join neighboring residues in a head-to-tail arrangement.
These connections between amino acids are rigid because of an electronic resonance that imparts
partial double bond character to the peptide bond.
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Most peptide bonds are in the trans configuration- Consequently, the atoms Cα-C-N'-Cα' are
confined to a plane in which Cα and Cα' may be in either a cis- or a trans- orientation with respect to
the peptide bond. Trans- peptide bonds are generally preferred, probably because of fewer steric
clashes between the side chain atoms of neighboring residues. However, cis- peptide bonds are
allowed amino-terminal to proline residues, owing to the unique cyclic side chain of proline (see below).
Another consequence of peptide bond resonance is the polarization of the amide nitrogen and the
carboxyl oxygen, creating a very good hydrogen bond donor and acceptor, respectively. Hydrogen
bonds between these main chain atoms are a prominent feature of many secondary structural motifs.
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Amino acids are chiral compounds- The peptide main chain is decorated with side chains that are
specified by the amino acid sequence. Each constellation of side chains confers unique physicochemical properties to a given peptide sequence. All amino acids except glycine have a chiral center
located at Cα. Naturally occurring amino acids are L-enantiomers, and this choice of hand defines
the region of conformational space that is accessible to a peptide chain. Proteins of identical sequence
consisting either of all L-amino acids or of all D-amino acids would be unable to adopt the same
structure because of stereospecific differences in the potential clashes between neighboring residues.
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Polypeptide conformation- Two dihedral angles, φ (phi) and ψ (psi), define the course of the
peptide chain. The φ angle designates rotation about the bond joining N to Cα, and ψ designates
rotation about the Cα-C bond. Rotation about these bonds causes crankshaft-like motions that alter
the course of the peptide chain, generating regular conformations like the β-strand, reverse turns, and
α-helices. The side chain attached to Cα of each amino acid limits rotation about φ and ψ because of
steric hindrance between the side chain and the adjacent main chain atoms. Glycine lacks a side chain
and is therefore more flexible than other amino acids. Thus, glycine is prevalent in turns, loops, and
other flexible segments of proteins. Regular secondary structures such as the α helix or the β-strand
have characteristic φ and ψ angles.
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The conformational space, or range of φ and ψ angles, of the residues within a protein can be displayed
on a Ramachandran plot (named for the biophysicist G.N. Ramachandran). Highly populated areas
on the Ramachandran plot correspond to residues within segments of regular secondary structure
(helices, sheets). Most of the remaining area of conformational space is forbidden to all residues except
glycine, because of steric clashes that would result from the main chain adopting these dihedral angles.
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Amino Acid Side Chains
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The 20 naturally occurring amino acids can be classified according to the properties of their side chains.
The side chain determines how a residue interacts with its neighbors and, to some degree, whether it is
likely to be located near the surface or buried within the protein fold.
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Aliphatic amino acids (A,V,L,I,M,P)- Hydrophobic amino acids dominate in the protein interior.
These are the most structurally diverse group of residues, reflecting their role in filling the irregular
spaces within the protein core.
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Aromatic amino acids (F, Y, and W)- Aromatic rings participate in π-electron stacking interactions
with one another and with ligands. The aromatic residues tyrosine and tryptophan, and to a lessor
extent phenylalanine, account for most of the near-UV range absorbance and fluorescence properties of
proteins. The spectral properties of these residues are strongly influenced by the local environment of
the aromatic ring, making them useful probes of protein structure.
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Polar amino acids (S, T, N, Q, and H)- Polar residues contain oxygen and/or nitrogen atoms that
participate in hydrogen bonds that stabilize various protein folds, assist chemical reactions, or participate
in the recognition of other molecules. The imidazole ring of histidine is a very effective nucleophilic
catalyst that is present in a number of enzyme active sites, including the catalytic triad of the serine.
Histidine is partially charged at physiological pH (pKa ≅ 6.2).
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Charged amino acids (D, E, K, and R)- these are typically located on the surface of proteins, where
they participate in long-range electrostatic interactions between protein subunits, or contacts with
substrates and other ligands. Examples include the active site aspartic acid in acid proteases (pepsin)
and the basic residues of DNA-binding domains.
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Glycine- Glycine lacks a side chain and is therefore achiral, being less sterically encumbered than the
other residues. Glycine can readily adopt conformations that are forbidden for other amino acids,
allowing irregular structures such as loops and turns at the protein surface.
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Proline, a special case - Proline is an imino acid with a side chain that forms a 5-membered
pyrrolidine ring. This severely restricts the range of the main chain dihedral angle φ and, consequently,
proline is greatly disfavored in certain secondary structures such as the α-helix. Prolines are typically
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found in loops and extend segments of the peptide chain.
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Cysteine and disulfide bonds- Cysteine is a polar amino acid that is readily deprotonated, forming a
highly reactive thiolate ion that can covalently bond with alkylhalides, heavy metals, or other cysteines.
A covalent link between 2 cysteine sulfurs is termed a disulfide bond. Disulfide bonds stabilize protein
secondary structure by crosslinking cysteine residues that are distant in the primary sequence. Disulfide
bonds are found in hormones (insulin), toxins, and some thermostabile proteins.
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Rotamers- Side chain conformations are described by the torsion angles χ1χ2, χ3 , etc. Energeticallyfavored conformations, termed side chain rotamers, have χ1 andχ2 angles of approximately -60 (g+),
+60 (g-), or 180 degrees. Most amino acids in high-resolution protein models conform to one of the
preferred rotamers.
Levels of Protein Structure
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Primary Structure - A protein's amino acid sequence determines its unique three-dimensional
structure. In general, proteins with homologous primary sequences adopt similar folds. However, the
amount of sequence relatedness that assures two proteins have homologous structures is difficult to
predict, and localized differences in structure can have important functional consequences. This means
that predictions of protein structure that are based on primary sequence data are tentative at best.
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Secondary Structure - Polypeptide chains fold into compact shapes that exclude water from the
protein interior. Segments of the peptide chain assume regular, repeating shapes that are determined by
favorable interactions between adjacent residues. These local packing arrangements typically involve
short (5 to 20 residues) segments and they are termed secondary structures. Common secondary
structures include α-helices, β-strands, and reverse turns. These structures are stabilized by repeating
patterns of hydrogen bonds between main chain oxygens and nitrogens.
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Tertiary and Quaternary Structure - Elements of secondary structure pack together in folded
proteins, bringing residues that are distal in the primary sequence into close proximity. Packing
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arrangements that involve segments of a single polypeptide chain are termed tertiary structure. Some
proteins aggregate, forming oligomers such as dimers, trimers, etc. Quaternary structure describes the
arrangement of subunits in oligomeric proteins.
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Larger proteins consist of discrete
modules of structure, termed
domains, that are connected by
extended segments. In many
cases, this “beads on a string”
modular architecture also
corresponds to discrete functional
modules. An example of this is the
Src protein kinase (right).
Principles of Protein Folds
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The interior of proteins is hydrophobic, consisting primarily of aliphatic residues that are in intimate
contact so as to exclude water. It is thought that exclusion of water from the hydrophobic protein
interior is one of the principle forces stabilizing the native structure of proteins. However, this places the
polar nitrogen and oxygen atoms of the protein main chain in a very hydrophobic environment in which
their hydrogen bonding potential must be satisfied. This problem has been solved by proteins folding
into regular, repeating secondary structures in which (almost) all main chain nitrogens and oxygens
participate in hydrogen bonds. Residues with charged or polar side chains are typically located on the
protein surface, where they participate in electrostatic interactions between neighboring domains, in the
binding of small ligands or in the recognition of other macromolecules.
Examples of Protein Secondary Structure
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α-Helices and helical bundles- The α-helix is one of the most common secondary structures. The
hand and pitch of an α-helix are determined by a repeating pattern of main chain dihedral angles
(approx. φ ≅ -60, ψ ≅ -40) that generate a regular helix. α-helices are right-handed, with about 3.6
residues per turn. This helical pitch means that residues along one face of the helix are located 3 to 4
residues apart in primary sequence. Some amino acids tend to be excluded from α-helices. Prolines
disrupt helices because they cannot adopt the preferred main chain dihedral angles, and because the
amide nitrogen is not available for hydrogen bonding with a nearby carbonyl (see below). Glycine tends
to destabilize α-helices because its conformation is less constrained and therefore less likely to adopt
the necessary backbone conformation.
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α-Helices are rigid, rod-like structures that are stabilized by a 'web' of hydrogen bonds between the
main chain amide nitrogens and carboxyl oxygens in adjacent turns of the helix. The carbonyl oxygen of
residue 'i' accepts a hydrogen bond from the amide nitrogen of residue 'i+4'. The dipoles associated
with these intrahelical hydrogen bonds are aligned, giving rise to a helix macrodipole with a partial
positive charge at the amino-terminus and a partial negative charge at the carboxyl-terminus of the helix.
Helix macrodipoles strongly contribute to the overall dipole moment of proteins and, in some instances,
this effect may guide or stabilize the interaction of proteins with charged molecules. For example, the
dipole moment of the 'recognition helix' of certain DNA-binding proteins is thought to contribute to the
stability of the protein-DNA complex.
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α-Helices are stabilized by a web of intrahelical hydrogen bonds between carbonyl oxygens and the
amide nitrogens of the residue located 4 residues (one turn) towards the C-terminus of the helix.
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Many helices are amphipathic; that is, they have a hydrophobic face and a hydrophilic face. This
amphipathic character facilitates packing against the hydrophobic protein interior while exposing the
opposite face of the helix to solvent. Helices may pack together, forming 2-, 3-, or 4-helix bundles that
are typically quite stabile and inflexible. Helical bundles are commonly seen at interfaces between
protein subunits, or in the filamentous proteins forming the cytoskeleton and other cellular structures.
Hydrophobic side chains facing the interior of the helical bundle pack tightly together, excluding water
from the interior of the folded protein. Additional stability is gained from hydrogen bonds and salt
bridges that link helices at the protein surface.
β-sheets- Another very common protein secondary structure is the β-sheet, which consists of a group of
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extended polypeptide chains termed β-strands. β-sheets are either parallel or anti-parallel, depending on
the orientation of adjacent strands. Both types of β-sheet are stabilized by hydrogen bonds between the
main chain atoms of adjacent strands, with all interior hydrogen bond donors and acceptors participating in
this network. In parallel sheets, all β-strands are oriented in the same N-terminal to C-terminal direction.
In anti-parallel sheets, adjacent strands have opposite polarities and the hydrogen bonds connecting
adjacent strands show an alternating pattern of wide and narrow spacing. The normal packing arrangement
between the strands of a β-sheet creates a slight right-handed curvature. Unlike α-helices, β-sheets
consist of peptide segments that may be distal in primary sequence, with the residues connecting the strands
comprising loops or other secondary structures.
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Loops and turns- Loops and turns connect the more regular elements of secondary structure. Reverse
turns are compact structures in which the peptide chain doubles back on itself, reversing the direction of
the main chain. Reverse turns are stabilized by hydrogen bonds bridging across the interior of the turn.
Loops are typically located on the protein surface and they include many of a protein's antigenic
epitopes. Loops and turns also serve as flexible hinges that allow movements between and within
protein domains.
Examples of Tertiary Structure - Motifs and Domains
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Motifs- Elements of secondary structure combine into simple folds termed motifs, which consist of a
limited number of α-helices and/or β-sheets and the intervening peptide segments. Examples of motifs
include the helix-turn-helix and the β-hairpin.
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Domains- A domain consists of several motifs packed in a specific, compact arrangement that in many
cases can fold as an independent unit. Small proteins may consist of a single domain, whereas larger
proteins typically consist of multiple domains connected by flexible segments of the peptide chain.
Domains are characteristically resistant to proteases, whereas the loops and strands connecting these
domains tend to be exposed and protease-sensitive. This feature can be exploited by cleaving a protein
into its constituent domains by limited proteolysis. In many cases, these isolated domains retain some of
the functions of the parent protein. For example, DNA-binding domains of many transcription factors
are independent modules that can be combined with other domains (such as a transcriptional activation
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domain) to create chimeric proteins with unique properties. This functional modularity is a direct
consequence of protein domain structure.
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Examples - simple motifs include the Greek key, which is a common topology linking the strands of
βsheets. The EF-hand motif (center) is a calcium-binding site formed at the junction of 2 α-helices.
The metal atom is chelated by acidic side chains and nearby main chain atoms. α-Helical coiled-coils
provide rigidity to structural proteins and to dimeric transcription factors like the basic region leucine
zipper proteins.
Forces Stabilizing the Native Folds of Proteins
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Genetic information is ultimately expressed in the production of proteins having unique activities and
structures that are specified by a particular amino acid sequence. How this linear genetic code is
converted into a precise three-dimensional protein structure remains one of the outstanding questions of
molecular biology, commonly known as the 'protein folding problem'.
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Proteins are only marginally stable at physiological temperature, and they are held together by a number
of weak, interdependent forces. The thermodynamic hypothesis of protein stability states that the
native conformation of a protein is thermodynamically the most stable conformation that a polypeptide
chain can assume. Therefore, the native conformation should not depend upon the particular folding
pathway to the native state nor require the presence of accessory proteins to guide the folding process
(molecular chaperones). The thermodynamic hypothesis also predicts that protein folding should be
spontaneous and reversible. These properties are not always observed experimentally; some proteins
require molecular chaperones to accelerate the folding rate to a biologically relevant time scale.
Weak, Noncovalent Interactions:
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Dispersion Forces - (0.01 - 0.2 kcal/mol) van der Waals interactions are isotropic (nondirectional)
and very weak. However, they are numerous and therefore are a major contributor to protein stability.
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Electrostatic Interactions
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Salt Bridges- (1-5 kcal/mol) weaker, long-range forces. Charged residues participating in salt bridges
are almost always at the protein surface. The strength of these interactions is influenced by pH, ionic
strength, and the local electrostatic environment.
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Hydrogen Bonds - (2 - 10 kcal/mol) Hydrogen bonds are directional
and therefore well suited for specifying particular interactions. Side
chains and main chain atoms participate in numerous hydrogen bonds at
the surface and in the interior of proteins. Examples include hydrogen
bonds between the turns of an αhelix or joining the strands of β-sheets.
Covalent Interactions
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Disulfide Bonds - Covalent bonds between two cysteine sulfur
atoms, termed disulfide bonds, are found in many proteins. These
disulfide bridges form spontaneously between specific cysteine pairs,
even under the reducing conditions within the cell. Disulfide bonds
provide added structural stability and many are involved in specific
functions. Although they are rare in comparison to the interactions
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discussed above, disulfide bonds are more abundant in proteins functioning in extracellular spaces, such
as digestive enzymes and peptide hormones.
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The hydrophobic effect- Arguably, the most important force stabilizing protein structure is the
hydrophobic force. It can be described as the aversion of aliphatic residues for the aqueous
environment of the cell, which causes hydrophobic residues to cluster within the interior of the folded
protein. Although the physical basis of the hydrophobic effect is complex, a striking feature (at
physiological temperatures) is the formation of ordered 'cages' of water around hydrophobic surfaces,
such as those of an unfolded polypeptide. The internalization of hydrophobic groups during protein
folding releases caged water, resulting in an entropy change that favors protein folding. Proteins
generally fold into compact shapes that exclude water from their interior. Typically, the van der Waals
radii of the atoms within a protein occupy 70% to 80% of a its volume. Thus, residues are very tightly
packed against one another in the protein interior.
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Events in protein folding- The acquisition of native structure is a highly cooperative process, which
approximates a two-state equilibrium between the unfolded and native conformations. Recent
improvements in proton exchange nuclear magnetic resonance techniques, and stopped flow circular
dichroism methodologies, as well as the development of peptide models of protein folding, have allowed
the characterization of partially-folded intermediates in the folding pathways of several proteins. These
studies suggest that different segments of the polypeptide chain fold independently and at different rates,
creating several regions of transient secondary structure.
Folding culminates with a coalescence of partially structured folding intermediates, creating the final native
structure. The thermodynamic hypothesis of protein stability postulates that the native structure is the most
stable conformation and it is not dependent on the particular pathway to the native state. Recent
experimental evidence suggests that a given protein may use several distinct folding pathways to achieve
native conformation. This mechanistic plurality may arise from conformational heterogeneity of the unfolded
polypeptide.
Most large proteins do not spontaneously fold on an experimentally observable time scale. These proteins
require the assistance of molecular chaperones, which guide the proper folding of proteins and multi-protein
complexes. The chaperones themselves assemble into oligomeric ribosome-sized, ring-shaped structures
that interact transiently with partially folded or denatured proteins, apparently without regard to the amino
acid sequence of the peptide chain. We will return to this topic later in the course.