Transcript Chapter 4A Lecture

Chap. 4A The Three-dimensional Structure
of Proteins
Topics
• Overview of Protein Structure
• Protein Secondary Structure
• Protein Tertiary and Quaternary Structure
• Protein Denaturation and Folding
Fig. 4-1. Structure of the enzyme
chymotrypsin, a globular protein
Overview of Protein Structure (I)
In principle, a protein could have a nearly limitless number of
shapes (structures) due to the fact that free rotation is allowed
about many of its covalent bonds. However, the great majority
of proteins have a specific chemical or structural function, which
suggests that each has a unique 3D structure. This idea is
supported by the finding that most proteins can be crystallized.
Nonetheless, most proteins display at least a moderate degree of
flexibility which is needed in their performance of function.
Interestingly, parts of many proteins have no fixed structure.
The lack of definable structure can be crucial to function.
Overview of Protein Structure (II)
The spatial arrangement of atoms in a protein or any part of a
protein is called its conformation. The functional conformation
of the protein is called its native state. The native state is
usually the conformation that is thermodynamically the most
stable. A protein’s conformation is stabilized largely by multiple
contributing weak noncovalent interactions. These include
hydrogen bonds, ionic interactions, van der Waals interactions,
and the hydrophobic effect of burying most nonpolar amino acid
side-chains in the interior of the protein. Disulfide bonds also
contribute to structural stabilization. In the native state, the
number of weak noncovalent interactions is maximal.
Peptide Bond Structure
X-ray diffraction studies of peptide bond structure by Linus
Pauling and Robert Corey revealed that the C-N peptide bond is
shorter than the C-N bonds in simple amines. Furthermore, the
six atoms that are part of the peptide group of a peptide bond
are coplanar. These data indicate that a resonance or partial
sharing of two electron pairs occurs between the carbonyl
oxygen and amide nitrogen (Fig. 4-2a). Because peptide bonds
have partial double bond character, they do not rotate freely.
The Backbone of a Polypeptide Chain
Rotation is permitted about the N-C (phi, ) and C-C (psi, )
bonds in the peptide backbone (Fig. 4-2b). Thus, the backbone of
a polypeptide chain can be pictured as a series of rigid planes
with consecutive planes sharing a common point of rotation at C.
The rigid peptide bonds limit the range of conformations possible
for a polypeptide chain. Note that the peptide bond occurs in a
trans configuration 99.6 % of the time. Cis peptide bonds are
very rare.
Ramachandran Plots of  and  Angles
Both the  and  angles are defined as being ± 180˚ when the
polypeptide backbone is fully extended and all peptide groups are in
the same plane (Figs. 2c & d). In principle,  and  can vary freely
between +180˚ and -180˚, but many values are prohibited by
steric interference between atoms in the polypeptide backbone and
amino acid side-chains. The conformation in which both  and  are
0˚ is prohibited for this reason. This conformation only serves as a
reference point for describing the dihedral angles between peptide
groups (planes). Allowed angles for  and  are shown in a
Ramachandran plot for poly L-alanine (Fig. 4-3).
Overview of Protein Secondary Structure
Secondary structure refers to stable, short-range, periodic
folding elements that are common in proteins. A regular
secondary structure occurs when each dihedral angle,  and ,
remains the same or nearly the same throughout the element.
There are a few types of secondary structures that occur widely
in proteins. These include the  helix, the b conformation, and ß
turns.
The  helix (I)
In the  helix (Fig. 4-4a), the
peptide backbone adopts a
cylindrical spiral structure in which
there are 3.6 amino acids per turn
(5.4 Å). The R groups point out
from the  helix axis, and mediate
contacts to other structure elements
in the folded protein. The  helix is
stabilized by hydrogen bonds
between backbone carbonyl oxygen
and amide nitrogen atoms that are
oriented parallel to the helix axis.
In fact the structure maximizes the
use of internal hydrogen bonding.
Hydrogen bonds occur between
residues located in the n and n + 4
positions relative to one another.
The  helix forms more readily than
many other conformations in part
because of its optimal use of
internal hydrogen bonding.
The  helix (II)
Other views of the  helix are shown in Figs. 4-4 b-d. Fig. 4-4 b
shows an end-on view of an  helix, and emphasizes how R groups
are located on the surface. A space filling model of an  helix is
shown is Fig. 4-4 c, and shows how there actually is no central
hole in the helix as seems to be the case using the ball-and-stick
model in Fig. 4-4 b. Fig. 4-4 d shows a helical wheel projection
of an  helix and emphasizes how the side chains of amino acids in
the n and n + 3 positions are in close contact. It also emphasizes
how different faces of the  helix can have different properties,
e.g., polarity.
 and  Angles for Secondary Structures
The backbone atoms of
amino acid residues in
the prototypical  helix
have characteristic
dihedral angles that
define the  helix
conformation (Table 41).  helical segments
in proteins often have
dihedral angles that
deviate slightly from
these ideal values. This
introduces slight bends
and kinks into the helix
axis.
 Helix Nomenclature
Protein structure studies have determined that right-handed 
helices occur in proteins (see figure in Box 4-1). Extended lefthanded  helices have not been observed in proteins, presumably
because they are theoretically less stable. Experiments have
shown that an  helix can form in a polypeptide consisting of
either L- or D-amino acids. However, all residues must be of one
stereoisomeric form or the other or the  helix will be disrupted.
The most stable form of an  helix formed by D-amino acids is
left-handed.
Worked Example 4-1. Secondary Structure
and Protein Dimensions
 Helix Stability and Amino Acid
Composition
Five types of constraints affect the stability of an  helix: 1) the
intrinsic propensity of an amino acid residue to form an  helix
(Table 4-2, next slide); 2) the interactions between R groups,
particularly those spaced three or four residues apart; 3) the
bulkiness of adjacent R groups; 4) the occurrence of proline and
glycine residues; and 5) interactions between amino acid residues
at the ends of a helical segment and the electric dipole inherent
to the  helix (Fig. 4-5, below).
 Helical Preferences of Amino Acids
The properties of the R group strongly affect the capacity of the
backbone atoms to take up the characteristic  and  angles of an
 helix (Table 4-2). Alanine with its small methyl group in its side
chain shows the greatest propensity to form an  helix under most
conditions. In contrast, amino acids such as threonine and
asparagine, with bulky groups attached to the ß carbon of the
amino acid show a reduced propensity to occur within an  helix.
Proline and glycine show little
tendency to occur in  helices. In
proline the N atom is part of a
rigid ring, and rotation about the
N-C bond is not possible. This
places a destabilizing kink into an
 helix. In addition, the N atom
when in a peptide bond linkage has
no substituent hydrogen that can
participate in hydrogen bonding to
other residues. Glycine is highly
conformationally flexible due to its
having a H atom for a side chain.
This is disruptive to the stability
of the  helix.
Helix Dipoles and  Helix Stabilization
A small electrical dipole exists in each
peptide bond. These dipoles are aligned
through the hydrogen bonds of the helix,
resulting in a net dipole along the helical
axis that increases with helix length (Fig.
4-5). For this reason, the helix is stabilized
when amino acids with negatively charged
side chains are located at the amino
terminus of the helix, and vice versa.
Placement of an amino acid with a positively
charged side chain near the amino terminus
of an  helix destabilizes it, as does the
placement of an amino acid with a negatively
charged side chain near the carboxyl
terminus of the helix.
ß Conformation
A second type of secondary structure, the ß conformation, is very
common in proteins. In this structure element, the polypeptide
backbone is nearly fully extended into a zigzag strand rather than
a helical structure (Fig. 4-6 a). The R groups of consecutive
amino acids in a ß strand are oriented on opposite sides of the
strand. As expected, the  and  angles for the ß conformation
are distinctly different from those observed in the  helix (Table
4-1).
Antiparallel and Parallel ß Sheets
The arrangement of several ß
strands side-by-side forms a
planar type structure called a ß
sheet (a.k.a., ß pleated sheet)
(Fig. 4-6 b & c). Hydrogen
bonds between adjacent strands
of the sheet stabilize the
structure. The adjacent
strands in a ß sheet can be
either parallel or antiparallel,
that is, having the same or
opposite N-to-C terminal
orientations, respectively . The
hydrogen-bonding patterns are
slightly different for the
antiparallel and parallel ß
sheets, with hydrogen bonds
being more perfectly aligned in
the former.
Structures of ß Turns
ß turns are common secondary structure elements that link
successive runs of  helix or ß conformation where the
polypeptide chain reverses direction in space (Fig. 4-7). ß turns
therefore, commonly are located at the surface of a globular
protein. Particularly common are ß turns that connect the ends
of two adjacent strands of an antiparallel ß sheet. The most
common types of ß turns (Type I and Type II ß turns) contain
four amino acids in which the first and fourth residues in the
turn are hydrogen bonded to one another. The second and third
residues in the ß turn commonly hydrogen bond to water at the
surface of the protein.
Proline Isomers
Glycine and proline often are present in ß turns. Glycine is
prevalent because its side chain is small, making the peptide
backbone quite flexible. Proline is common because peptide bonds
involving the imino nitrogen of proline readily assume the cis
configuration (Fig. 4-8). For peptide bonds involving the imino
nitrogen of proline, about 6% are in the cis configuration and
many of these occur in ß turns.
Ramachandran Plots of
Secondary Structures
The idealized dihedral angles that
define the  helix and ß
conformation fall within relatively
restricted regions of sterically
allowed structures on a
Ramachandran plot (Fig. 4-9a). Most
experimentally determined values of
 and  measured in known protein
structures fall into these regions,
with high concentrations near the 
helix and ß conformation values as
predicted (Fig. 4-9b).
Overview of Tertiary and Quaternary
Structure
Tertiary structure refers to the overall three-dimensional
arrangement of all atoms in a protein. Tertiary structure deals
with long-range aspects of the fold of a protein, including
interactions that form between isolated elements of secondary
structure. Both noncovalent and covalent interactions are
included in the tertiary structure. Quaternary structure refers
to the contacts between, and overall arrangement in threedimensional space of the individual subunits of a multisubunit
protein.
Fibrous and Globular Proteins
Many proteins can be classified into two groups based on their
general structural features. Fibrous proteins, such as keratins and collagens, have polypeptide chains arranged in long
strands or sheets. These proteins usually consist mostly of a
single type of secondary structure and their tertiary structure
is relatively simple. Globular proteins, such as myoglobin and
serum albumin, consist of polypeptide chains that are folded
into a spherical or globular shape. Globular proteins often
contain several types of secondary structure. Fibrous proteins
generally function to provide support, shape, and external
protection to vertebrates; globular proteins often function as
regulatory proteins and enzymes. The dimensions of globular
proteins (e.g., human serum albumin, 585 aas) are much
smaller than they would be if their chains were exclusively 
helical or adopted the ß conformation (Fig. 4-15).
Examples of Fibrous Proteins
The properties of the fibrous proteins we will discuss are
summarized in Table 4-3. All of these proteins are insoluble in
water, and they contain a high proportion of nonpolar amino acid
residues both in their interiors and on their surfaces. The
hydrophobic surfaces are largely buried because many similar
polypeptide chains are packed together to form elaborate
supramolecular complexes.
Fibrous Proteins: -Keratins (I)
The -keratins, which are only found
in mammals, constitute most of the
dry weight of hair, wool, nails, claws,
quills, horns, hooves, and much of the
outer layer of skin. Hair -keratin
(Fig. 4-11) consists of an elongated
right-handed  helix with somewhat
thicker structures near the N- and
C-termini. Pairs of -keratin
monomers interwind in a left-handed
sense, forming two-chain coiled coils.
These then combine in higher order
structures called protofilaments,
protofibrils, and intermediate
filaments. Intermediate filaments
contain 32 monomeric strands of keratin. -keratin is rich in the
hydrophobic amino acids Ala, Val,
Leu, Met, and Phe. -keratins are
strengthened by covalent disulfide
bond cross-links between monomers in
the coiled-coil and higher order
structures. In rhinoceros horn keratin, ~18% of the residues are
cysteines involved in disulfide bonds.
Fibrous Proteins: -Keratins (II)
The -keratins of hair lengthen when exposed to moist heat. This
occurs due to penetration of water into the  helices of hair
fibers where it competes with intrachain hydrogen bonds. The
hydrated, stretched -keratins adopt a more ß conformation-like
structure. Hair -keratins are disulfide bond-linked, and
consecutive reduction and oxidation steps are used in the process
of waving and curling hair (permanents, figure in Box 4-2).
Fibrous Proteins: Collagen
Collagen is a structural protein found in connective tissue such as
tendons, cartilage, the organic matrix of bone, and the cornea of
the eye. The collagen helix has a unique secondary structure which
is left-handed and has three amino acids per turn (Fig. 4-12 a &
b). Three collagen polypeptides ( chains) associate in a righthanded superhelical coiled coil structure called the three-stranded
collagen superhelix (Fig. 4-12c & d). Vertebrate collagen
typically contains about 35%
Gly, 11% Ala, and 21% Pro and
4-hydroxyproline. The Pro and
4-Hyp residues permit the
sharp twisting of the collagen
helix. The repeating tripeptide
Gly-X-Y, where X is often Pro
and Y is often 4-Hyp, occurs
frequently in the  chain
sequence. Gly residues are
located where the three chains
of the collagen superhelix
contact one another (Fig. 4-12
d, red).
Structure of Collagen Fibrils (I)
Collagen (Mr 300,000) is a rod-shaped
molecule about 3,000 Å long and only
15 Å thick. Its three helically
intertwined  chains can have different
sequences and each chain has about
1,000 amino acid residues. Collagen
fibrils are made up of collagen
molecules aligned in a staggered fashion
and cross-linked for strength. The
specific alignment and degree of crosslinking vary with the tissue and produce
characteristic cross-striations in an
electron micrograph (Fig. 4-13, upper).
A typical mammal expresses on the
order of 30 structural variants of
collagen. Diseases such as osteogenesis
imperfecta and Ehlers-Danlos syndrome
are caused by mutant alleles of collagen
genes. Commonly an amino acid with a
relatively large R group such as Cys or
Ser replaces Gly residues in mutant
collagens, disrupting their structure and
function.
Structure of Collagen Fibrils (II)
The individual  chains of triple-helical collagen molecules and
the collagen molecules of fibrils are cross-linked by covalent
bonds involving lysine, 5-hydroxylysine, and histidine residues
that are present at some of the X and Y sites in the triplet
repeat. The dehydrohydroxylysinonorleucine residue produced by
cross-linking between lysine and 5-hydroxylysine residues is
shown below. With aging, cross-linking frequency increases
contributing to the brittle properties of aging connective tissue.
Synthesis of 4-hydroxyproline
As noted above,collagen chains are constructed of the repeating
tripeptide unit Gly-X-Y, where X often is Pro and Y often is 4Hyp. The proline ring normally occurs as a mixture of two
puckered conformations, called C-endo and C-exo (see below).
The collagen helix requires that the Pro residue in the Y positions
of the repeat adopt the C-exo conformation. This conformation
is enforced by the 4-hydroxyl group of 4-Hyp, and this is why
this amino acid occurs in collagen chains. In the absence of
vitamin C, cells cannot hydroxylate the Pro in the Y positions.
This leads to collagen instability and the connective tissue
problems seen in the deficiency disease known as scurvy. 4-Hyp
is synthesized by the enzyme prolyl 4-hydroxylase.
Vitamin C and Scurvy (I)
Vitamin C (L-ascorbic acid) plays an important role in the
production of collagen. In vitamin C deficiency (scurvy),
connective tissue synthesis and function are impaired (Box 4-3).
In extreme deficiency cases, the individual experiences numerous
small hemorrhages caused by fragile blood vessels, tooth loss,
poor wound healing and the reopening of old wounds, bone pain
and degeneration, and eventually heart failure. Vitamin C is
important in maintaining the enzyme prolyl 4-hydroxylase in its
active state. In the normal prolyl 4-hydroxylase reaction (see
below), one molecule of -ketoglutarate and one O2 bind the
enzyme along with a Pro residue. The -ketoglutarate is
oxidatively decarboxylated to form succinate and CO2, and the
remaining oxygen atom is used to hydroxylate the appropriate
Pro residue in procollagen. L-ascorbate is not needed in this
reaction.
Vitamin C and Scurvy (II)
L-ascorbic acid plays a vital role in preventing prolyl 4hydroxylase from being inactivated in a second reaction that it
commonly catalyzes. In this reaction (see below), ketoglutarate is oxidatively decarboxylated to succinate and CO2
in a reaction that does not involve hydroxylation of proline. In
this reaction a critical Fe2+ metal ion of the enzyme is oxidized
to the Fe3+ state, inactivating the enzyme for further catalysis.
When L-ascorbic acid is present, the iron atom is reduced to the
Fe2+ state, maintaining the active form of the enzyme.