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Chapter 4 2
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Protein Secondary Structure Overview of Protein Structure
Long chains of amino acids will commonly fold or curl Efforts of Linus Pauling & Robert Corey
into a regular repeating structure • Pauling, Corey and collaborators used X-ray diffraction to study the
structure of fibrous proteins (α-keratin and β-keratin)
– They identified two repeating patterns: (1) A 0.55 nm pattern in α-keratin and
(2) A 1.3 – 1.4 nm pattern in β-keratin
• Pauling also studied structures of small molecules containing amide
bonds
• From these studies, they proposed several principles that a protein
structure must obey:
– The bond lengths and bond angles should be distorted as little as possible
– No two atoms should approach one another more closely than is allowed by
their van der Waals radii
– The amide group must remain planar and in the trans configuration (this
allows for rotation only about the two bonds adjacent to the Cα)
– Some kind of non-covalent bonding is necessary to stabilize a regular folding
pattern
Chapter 4 5 Chapter 4 6
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Overview of Protein Structure Overview of Protein Structure
The Peptide Bond is Rigid and Planar The Peptide Bond is Rigid and Planar
Conformation of the Peptide Backbone is Determined by • In principle, phi and psi can have any value between -180° and +180°
the Phi and Psi Rotational Bond Angles • However, there are many combinations of angles that are prohibited due
Φ (phi) is the N – Cα bond (hetero)
to steric interference between:
– atoms in the peptide backbone φ=0
– the AA side chains
• For the backbone: and
– is usually between –45° and –180°
ψ=0
– Psi is usually between –60° and +190°
is forbidden
– For proline, Phi is limited to –35° to –85°
• For the R Group:
Ψ (psi) is the Cα – C bond (hetero) – Think about the structure of the R Group The allowed values for phi and psi
are shown graphically when psi is
– Compare glycine and alanine plotted versus phi in a
• By convention, both phi and psi are defined as 180° when the polypeptide
is in its fully extended conformation and all groups are in the same plane Ramachandran Plot
Chapter 4 9 Chapter 4 10
Fully allowed
Fully allowed
At limits of
allowability
(not Val, Ile)
At limits of
allowability Allowed with
bond flexibility
Pro
Chapter 4 11 Chapter 4 12
Fig. 7-9 in Voet & Voet, 1990
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Overview of Protein Structure Protein Secondary Structure
The Peptide Bond is Rigid and Planar
A Structurally Confined AA – Proline
• Protein Secondary Structure can be broken
into three main types:
– Helical Structure
Φ (phi) is limited – Sheet Structure
– Connective Structure
• These types vary in their size, hydrogen
bonding patterns and positioning of the R
groups.
Pro • In some cases, structure can select for or
against a particular Amino Acid R group.
Chapter 4 13 Chapter 4 14
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Protein Secondary Structure Protein Secondary Structure
Which may form a two-stranded secondary The β-Sheet
structure called a β-ribbon • β-sheets are composed of
extended polypeptide chains in
• In which hydrogen bonds form straight pleated sheets
across between the two strands, NH---O=C • The hydrogen bonds formed are
interstrand rather than
• Where adjacent pairs of AA sidechains intrastrand.
alternate projecting above and below the • The pleat is the result of the
tetrahedral nature of the Cα
plane of the β-ribbon followed by the planarity of the
peptide bond.
• Which has a slight right-hand twist • The R groups are perpendicular
• And which if continued, becomes a multi- to the peptide backbone sheet
and alternate (up and down) from
stranded antiparallel β-sheet face to face.
Molecular Biology of the Cell, Alberts et al., 2002
Chapter 4 17 Chapter 4 18
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Protein Secondary Structure Protein Secondary Structure
Top View Parallel β-Sheet
Note:
• H-bonds distorted
•
•
R-group location
Pleated character
Connections
•
•
R1-R3 distance
Connected by RH Between
Parallel
crossover
• Usually has more than 5
strands
0.65 nm
• Avg. strand length is about
6 AA’s
β-Strands
Side View
Chapter 4 21 Chapter 4 22
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The α-Helix
Protein Secondary Structure C-terminus
The α-helix Cα
Cα
• A helix is characterized by the number (n) of
peptide units per helical turn and its pitch (p),
which is the distance the helix rises along its
• Most common (about 25% of the amino Cα
axis per turn
• The alpha helix has a pitch of 5.4 Å and 3.6
acids in proteins are in this structure) 3.6 residues
residues per turn
5.4Å pitch Cα • There are 13 atoms along the backbone per
• Stability is greatly enhanced by internal van 13
Cα
turn. This standard helical conformation is
called a 3.613 helix
der Waals contacts
Cα
• The hydrogen bonds of the helix are arranged
Cα 10 so that the peptide carbonyl bond of the nth
12 9
• H-bonds are in-line, optimum distance 11 8 Cα residue points along the helix towards the
peptide N-H bond of the (n + 4)th residue
5
• R-groups project outward, and provide the 4
Cα 6
7 • The core of the alpha helix is tightly packed
with a diameter of about 6Å. This packing
Cα
main constraints on helical structure 2
3 allows the interior atoms to be in van der Waals
contact across the helix thereby maximizing
N-terminus 1
their association energies.
• Amino acid residues in an alpha helix have conformations with ψ = -60° and φ = - 45 to
- 50°. This combination of angles prevents steric repulsion from the R groups
Chapter 4 25 Chapter 4 26
Chapter 4 27 Chapter 4 28
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Protein Secondary Structure Protein Secondary Structure
R-Group Constraints on α-Helical Structure The Helix Dipole
• Each carbonyl of the helix is hydrogen
• Interactions between AA side bonded to the peptide amide proton four
Arg
chains can stabilize or destabilize residues farther up the chain.
a helical structure • All hydrogen bonds are parallel to the helix
– Proline is a “helix breaker” (WHY?) axis.
– Adjacent residues of like charge • All carbonyl oxygens point in one direction,
Asp while all of the amide protons point in the
prevent helix formation (by repulsion
and H-bond destabilization) opposite direction.
– High glycine content favors alternate • This gives the helix a macroscopic dipole
structures instead moment from the individual dipoles.
The π Helix
• The π helix is a 4.416 helix.
• It has a mildly forbidden conformation and
has only been observed at the ends of a few
α-helicies.
• It has a comparably wide and flat
conformation which is too small to admit
water molecules but too large for van der
Waals contacts of the interior atoms across
the helical axis.
Chapter 4 31 Chapter 4 32
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Protein Secondary Structure Overview of Protein Structure
Ramachandran Revisited Amino Acid Sequence Determines 2° Structure of Proteins
A protein “knows” what its active conformation is!
Ramachandran Plot for
Pyruvate Kinase • Christian Anfinson conducted a denaturation
experiment to demonstrate this point.
• In 1957, he showed that ribonuclease A (124
AA’s), after having been completely denatured
using 8 M urea and 2-mercaptoethanol, regained
full enzymatic activity when the urea and 2-ME
were slowly removed by dialysis.
• Conclusion: the protein “knows” how to re-fold into
its active configuration.
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
3° Structure describes the folding of a protein’s 2° structure units together
Fibrous versus Globular Proteins
with the spatial dispositions of its side chains • Once we reach the 3° level, we can classify proteins into
This type of structure results from the interaction of two groups, fibrous or globular
the side chains and the peptide backbone
• Fibrous proteins have polypeptide chains arranged into
Possible interactions include: long strands or sheets
– Consist largely of a single type of 2° structure
Electrostatic Forces
Covalent bonding – Usually proteins that provide support and structure
Hydrophobic Forces • Globular proteins have polypeptide chains folded into
Non-covalent Interactions spherical or globular shapes
– Consists of several types of 2° structure
Proteins will select a
folding conformation that – Usually an enzyme or regulatory protein
gives the lowest free
energy
Chapter 4 37 Chapter 4 38
Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
The Remarkable Fibrous Proteins Hair is Made of α-Keratin
Right-hand
Left-hand
• insoluble in water (lots of hydrophobic residues) • The double coiled-coil amplifies the strength of the α-helix, like rope
• involved in structural roles within the cell • Hydrophobic R-groups (Ala, Val, Leu, Ile, Met, and Phe) interlock in a regular
pattern where the helices touch
• Covalent interchain disulfide links further stabilize keratins (rhinoceros horn is 18%
cysteine in S-S crosslinks)
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Some Helices Can Be Left-Handed! Connective Tissues are made of Collagen
• Proline is bad for right-handed helices, because of its φ / ψ angles and
its inability to form H-bonds (N is bonded to 3 carbons already)
• Left-hand single helix, 3 residues
per turn
• Polyproline, however, can form a left-handed helix with 3 residues per
turn, and a pitch of 9Å • 35% Gly, 11% Ala, 21% Pro / 4-
• Modified proline (4-hydroxyproline) allows the formation of hydrogen HydroxyP
bonds, and further stabilization of this unusual helical form
• Triplet Gly-X-Pro (or Gly-X-HyP)
• This post-translational modification requires vitamin C as a co-
substrate for 3 enzymes that are involved in the hydroxylation of both
repeats
proline and lysine • Supertwisted coiled coil is right-
handed, made of 3 left-handed α-
chains
Why do we care?
Stronger than steel!
Chapter 4 41 Chapter 4 LH RH 42
Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Collagen – End View of Triple Helix Collagen Fibrils Are Built From the Triple Superhelices
• The monomer is ~ 1000 AA
What effect would a • In the triplex, each may be
point mutation in a different in sequence
collagen monomer • Crosslinking of the triplexes (via
Gly unusual AA-links) gives more
have…? strength (but also more
brittleness, with age)
• Alignment is also tissue-specific,
and may vary in cartilage, bone
matrix, tooth dentin, skin,
tendon, and connective tissue
(e.g. lungs)
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Silk Fibroin: Antiparallel β-Sheet Stacking Globular Proteins are the Functional Workhorses of the Cell
• Functional variety reflects the variety of structural elements used to
• High tensile strength, construct them
but flexible – Includes Enzymes, Transporters, Motor proteins, Regulatory proteins,
• Mostly close-packed β- Immunoglobulins and Capsid proteins
• Because they are roughly spherical, there are lots of bends, loops,
sheets and folds
• Rich in Ala and Gly • These proteins are compact and comprise regions of α-helix, β-
(alternating – why?) sheets, β-turns, and others as well
• Protein stability results from “hydrophobic” core and disulfide bridges
• No covalent bonds • Structural motifs tend to be reused when similar functions are required
between strands or by the cell
sheets which gives the
material flexibility
Chapter 4 45 Chapter 4 46
Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
What General Principles Govern the Structures of One Nice, Simple, Globular Protein is Myoglobin
Globular Proteins? • A small protein (153 AA’s, 16.7 kD)
• Great stability is derived from internal hydrophobic • With a heme prosthetic group, so it can
interactions (very close packing allows van der • Bind oxygen in muscle cells
Waals forces to contribute strongly) • It is mostly α-helix (78% of its AA’s are in
• Polar groups are usually hydrated, and most are the 8 segments, from 7 to 23 AA’s long),
linked by turns (some of them β)
on the surface of the protein
• Much of its stability comes from
• Charged groups are likewise usually on the hydrophobic interactions
surface of the protein
• Peptide bonds are mostly trans, and Pro residues
are found at bends
• Smaller proteins often use disulfide linkages to
compensate for lower vol:surface area ratios
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Supersecondary Structures: All Alpha Supersecondary Structures: All Beta
β sandwich:
Helix-turn-Helix: Retinol Binding Protein
RNA-binding Protien Rop • Composed of two
• Two Helicies lie anti- separate sheets
parallel to one another and • Can be aligned or
are connected by a short orthogonal
loop
β Barrel:
Globin Fold: Four Helix Bundle: Porin
Myoglobin Cytochrome b562 • Sheets wrap around
• Consists of 8 helicies • Helicies are part of a β Helix:
to form a complex
• The two at the end of single peptide chain Pectate Lyase
with a central core
the chain are anti- and connected to each • Excellent for trans- • New fold recently
parallel, forming a other by three loops discovered
membrane proteins
helix-turn-helix fold • Sheet wind around in
like porin
a helical structure
Chapter 4 49 Chapter 4 50
Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Supersecondary Structures: Alpha / Beta Peptide Domains
Substrate Binding
Domain
Large Polypeptides (> 200 residues)
usually fold into two or more globular
clusters called Domains
Substrate Binding
Segregation of Functions
α/β Horseshoe: Multi-functional Proteins
α/β Barrel:
Ribonuclease Inhibitor Domain Repeats
Phosphate Isomerase
• Does not form a barrel NAD-Binding
Spacer Domains
• Here, the sheets are Domain
because the sheet are
slanted nearly parallel D- glyceraldehyde-
nearly parallel to the
to the central axis due 3-phosphate dehydrogenase
central axis
to the twist of the
sheet.
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Many proteins are not single peptide strands Symmetry in Oligomeric Structures
Instead, they are a combination of several distinct • Symmetry may be necessary to enhance function
peptide chains which function together as a unit
(can you think of examples…?)
• Types of symmetry include:
– Rotational (cyclic) – Cn
– Dihedral – Dn
– Helical
Hemoglobin
Chapter 4 53 Chapter 4 54
21-mer of Ferritin Single Subunit of Ferritin
Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Rotational Superposition Multiple Symmetry Axes
All subunits can be related by rotation about one or both of two axis,
Subunits are related by rotation about q single n-fold axis, one of which is two-fold
where n is the number of subunits
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Protein Tertiary & Quarternary Structure Protein Tertiary & Quarternary Structure
Larger Spherical Structures May Require More Helical Symmetry is “Open” and Long
Complex Symmetry Structures are Possible
Icosa = 20
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Protein Denaturation and Folding Protein Denaturation and Folding
The Thermodynamic “Free-Energy Funnel” of So in the folding of a protein…
Protein Folding
• There are two basic rules (after the secondary
structures are formed)
But in the cell, kinetics – Pack as close together as possible (why?)
may also contribute to – Minimize contacts between hydrophobic groups and
finding the correct water
conformation…why?
• The average contribution to stability is only about
0.2-0.4 kJ/mole for each amino acid
– Totaling ~ 40 kJ/mole for a peptide of 100 AA’s (or only
about 2 hydrogen bonds…)
– So proteins are also rather susceptible to denaturation
(unfolding)
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Protein Denaturation and Folding Protein Denaturation and Folding
Protein Renaturation Molecular Chaperones
• Molecular Chaperones are proteins that interact with partially folded or
• Many proteins can re-nature and
improperly folded polypeptides, facilitating correct folding
regain full function following mild • Heat Shock Protein 70 (Hsp70) and associated proteins
denaturation (what would – Bind unfolded regions to prevent aggregation
constitute harsh denaturation?) – Similarly facilitate folding during biosynthesis
• Disulfide linkages can re-form if – Block folding of some proteins prior to translocation to the correct
reducing agents are removed cellular compartment
• Chaperonins
• This phenomenon shows that
– Huge heptameric complexes (GroEL and GroES)
primary structure contains all the
– Required for folding of 10-15% of E. coli proteins
information necessary to
• Protein disulfide isomerase (PDI) and peptide prolyl cis-trans
reconstitute 2o, 3o, and 4o isomerase
structures – Shuffle disulfide cross-links to the correct conformation
– Interconvert proline peptide bonds that hinder folding
Chapter 4 65 Chapter 4 66
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