Proteins

the primary biological macromolecules of

living organisms
Protein structure and folding
Primary – Secondary – Tertiary – Quaternary
structure of proteins
 Structure of Proteins
• Protein molecules adopt a specific 3-dimensional
conformation in the aqueous solution

• This structure is able to fulfill a specific biological function

• This structure is called the native fold

• The native fold has a large number of interactions within the

protein

• There is a cost in conformational entropy during folding of

the protein into one specific native fold
A simulated folding pathway
 Interactions in Proteins

• Hydrophobic effect

• Hydrogen bonds

• London dispersion

• Electrostatic interactions
Structure of the Peptide Bond
• Structure of the protein is partially dictated
by the properties of the peptide bond
 The planar peptide bond
• Each peptide bond has some (~ 40%) double-bond
character due to resonance and cannot rotate.

The peptide bond is a resonance hybrid of two canonical structures

Structure of the Peptide Bond

• The resonance causes the peptide bonds

– be less reactive compared to e.g. esters
– be quite rigid and nearly planar
– exhibit large dipole moment
 The Rigid Peptide Plane and
the Partially Free Rotations

• Rotation around the peptide

bond is not permitted

• Rotation around bonds

connected to the alpha
carbon is permitted
 The Rigid Peptide Plane and
the Partially Free Rotations
• Φ (phi): angle around the -
carbon—amide nitrogen bond
(C-Cα-N-C)

• y (psi): angle around the -

carbon—carbonyl carbon
bond (N-C-Cα-N)

• In fully extended polypeptide,

both y and f are 180°
 Distribution of f and y
Dihedral Angles
• Some f and y combinations are very unfavorable because
of steric crowding of backbone atoms with other atoms in
the backbone or side-chains

• Some f and y combinations are more favorable because of

chance to form favorable H-bonding interactions along the
backbone

• Ramachandran plot shows the distribution of f and y

dihedral angles that are found in a protein

• shows the common secondary structure elements

• reveals regions with unusual backbone structure

Ramachandran Plot
Ramachandran Plot
 Secondary Structures
• Secondary structure refers to a local spatial
arrangement of the polypeptide chain
• Two regular arrangements are most common:
• The  helix
– stabilized by hydrogen bonds between nearby
residues
• The  sheet
– stabilized by hydrogen bonds between adjacent
segments that may not be nearby
• Irregular arrangement of the polypeptide chain is
called the random coil
The  helix
 The  helix
• Right-handed helix with 3.6 residues
(5.4 Å) per turn

• Helical backbone is held together by

hydrogen bonds between the nearby
backbone amides

• Side chains point out and are roughly

perpendicular with the helical axis
 The  helix: Top View

• The inner diameter of the helix

(no side-chains) is about 4 – 5 Å
• Too small for anything to fit
“inside”

• The outer diameter of the helix

(with side chains) is 10 – 12 Å
• Happens to fit well into the
major groove of dsDNA
 Sequence Affects Helix Stability
• Not all polypeptide sequences adopt -helical
structures

• Small hydrophobic residues such as Ala and

Leu are strong helix formers

• Pro acts as a helix breaker because the

rotation around the N-Ca bond is impossible

• Gly acts as a helix breaker because the tiny R-

group (hydrogen) allows other conformations
 Sheets
  Sheets
• The backbone is more extended with
dihedral angles in the range of ( 90 <
y < 180)
  Sheets
• The planarity of the peptide bond and
tetrahedral geometry of the -carbon create
a pleated sheet-like structure
• Sheet-like arrangement of backbone is held
together by hydrogen bonds between the
more distal backbone amides
• Side chains protrude from the sheet
alternating in up and down direction
Parallel and Antiparallel  Sheets
• In parallel  sheets the H-bonded strands run in
the same direction
Parallel and Antiparallel  Sheets

• In antiparallel  sheets the H-bonded strands

run in opposite directions
  Turns
• -turns occur frequently whenever strands in 
sheets change the direction
• The 180° turn is accomplished over four amino
acids
• The turn is stabilized by a hydrogen bond from a
carbonyl oxygen to amide proton three residues
down the sequence
• Proline in position 2 or glycine in position 3 are
common in -turns
Structures of β turns
 Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a polypeptide chain or in a
protein
• One can distinguish two major classes
– fibrous proteins
¤ typically insoluble; made from a single
secondary structure
– globular proteins
¤ water-soluble globular proteins
¤ lipid-soluble membraneous proteins
Structure of collagen
fibrous protein
Structure of whale myoglobin
globular protein
An ABC transporter of E. coli
globular membrane protein
Motifs (folds) - Protein Folding Patterns
Common arrangements of several
secondary structure elements
 How many folds?

• The number of unique folds in nature is fairly small (possibly a few

thousands)
• 90% of new structures submitted to PDB in the past decade have similar
structural folds in PDB, practically no new folds in the last three years
 Quaternary Structure
• Quaternary structure is formed by spontaneous
assembly of individual polypeptide subunits into a
larger functional cluster
Dimer

Cro protein of
bacteriophage lambda
two identical subunits

Tetramer

Human hemoglobin

two alpha(red)
two beta(yellow) subunits
4 heme groups
Primary – Secondary – Tertiary – Quaternary structure
Summary

(a) Linear Sequence of amino acids.

(b) Local folding into specific peptide

backbone conformations. Stabilized by h-
bonding and other non-covalent
interactions between atoms in peptide
backbone.

(c) Final folded 3-dimensional structure of a

single polypeptide chain. Stabilized by non-
covalent interactions between amino acid
side chain residues.

(d) Specific aggregation of two or more

polypeptide chains. Often characterized by
its symmetry.
 Protein Stability and Folding
• A protein’s function depends on its three-
dimensional structure.
• Loss of structural integrity with accompanying loss
of activity is called denaturation
• Proteins can be denatured by
• heat or cold
• pH extremes
• organic solvents
• chaotropic agents: urea and guanidinium
hydrochloride
• reduction of disulfide bonds by Mercaptoethanol
Renaturation of unfolded, denatured protein
ribonuclease
Ribonuclease Refolding Experiment
• Ribonuclease is a small protein that contains 8 cysteins
linked via four disulfide bonds
• Urea in the presence of 2-mercaptoethanol fully
denatures ribonuclease
• When urea and 2-mercaptoethanol are removed, the
protein spontaneously refolds, and the correct disulfide
bonds are reformed
• The sequence alone determines the native conformation
• Quite “simple” experiment, but so important it earned
Chris Anfinsen the 1972 Chemistry Nobel Prize
How Can Proteins Fold So Fast?

• Proteins fold to the lowest-energy fold in the microsecond

to second time scales. How can they find the right fold so
fast?

• It is mathematically impossible for protein folding to occur

by randomly trying every conformation until the lowest
energy one is found (Levinthal’s paradox)
 Levinthal’s paradox:
There are approximately 1050 possible
conformations for a typical protein
(~125 amino acids). Even if it took only
10-13 sec to try out each conformation,
it would take 1030 years to try a
significant fraction of them.
Obviously
folding does not happen randomly
direction toward the native structure is
thermodynamically most favorable
The thermodynamics of protein folding
depicted as a free-energy funnel
 Chaperonins

• Special class of molecular chaperones

that facilitate protein folding
• About 10 to 15% of proteins in E. coli
require chaperonins to right folding
• Require ATP
 Other examples of assisted molecular
processes in folding and posttranslational
modification of proteins

• Disulfide crosslinking bonding – disulfide

isomerase
• cis – trans isomerization of proline – prolyl cis-
trans isomerase
• Hydroxylation of Proline in collagen – prolyl 4-
hydroxylase (ascorbate demand)
Protein function
Functions of Globular Proteins
• Storage of ions and molecules
– myoglobin, ferritin
• Transport of ions and molecules
– hemoglobin, serotonin transporter
• Defense against pathogens
– antibodies, cytokines
• Muscle contraction
– actin, myosin
• Biological catalysis - enzymes
– chymotrypsin, lysozyme
 Example of Protein Function
and Ligand Binding

Myoglobin
• single polypeptide
• oxygen storage in tissues, muscle

Hemoglobin
• tetramer of two alpha and two beta subunits
• transports oxygen from lungs to tissues
 Ligand Binding

• Binding - reversible, transient process of chemical

equilibrium: A + B 
 AB

• A molecule that binds is called a ligand (typically a

small molecule)
• A region in the protein where the ligand binds is
called the binding site
• Ligand binds via non-covalent forces, which
enables the interactions to be transient
 Function of Myoglobin
• Organisms need to store oxygen for metabolism
• Generaly - protein side-chains lack affinity for O2

• Heme - Fe2+ in free heme could be oxidized to Fe3+

• Heme is bound to protein, Fe3+ is protected by a
His residue

• In mammals, myoglobin is the main oxygen

storage protein
Structures of Porphyrin and Heme
Structure of Myoglobin
 Binding of Carbon Monoxide
• CO has similar size and shape to O2; it can fit to the same
binding site
• CO binds to heme over 20,000 times better than O2
because the carbon in CO can be donated a lone electron
pair to vacant d-orbitals on the Fe2+
• Protein pocket decreases affinity for CO, but is still binds
about 250 times better than oxygen
• CO is highly toxic as it competes with oxygen. It blocks the
function of myoglobin, hemoglobin, and mitochondrial
cytochromes that are involved in oxidative phosphorylation
 Example of a Binding Pocket
O2 and CO in hemoglobin and myoglobin
 Could Myoglobin Work as
Good O2 Transporter?

• pO2 in lungs is about

13 kPa: it sure binds
oxygen well

• pO2 in tissues is about

4 kPa:

it will not release it!

 Simple change in the Cooperation of two binding
affinity would help but is (affinity) states is indeed
not the ideal solution better solution
 For Effective Transport
Affinity Must Vary with pO2

• pO2 in lungs is about 13 kPa: it sure binds

oxygen well

• pO2 in tissues is about 4 kPa: it releases about

half of it at pH 7.6
 How Can Affinity to Oxygen
Change Like This?

• Must be a protein with multiple binding sites

• Binding sites must be able to interact with
each other
• This phenomenon is called cooperativity

Positive cooperativity can be recognized by

sigmoidal binding curves
 Hemoglobin
• Hemoglobin is a tetramer of two different
subunits (22)
• Each subunit is similar to myoglobin
 Hemoglobin
Binding sites must be able to interact with each other
 Subunit Interactions: Details
Binding sites must be able to interact with each other

Example of some
interactions

Interactions can be
between subunits and also
within one subunit
 Conformational Change is
Triggered by Oxygen Binding
Hemoglobin is an allosteric protein

• allosteric proteins change their activity due to

induced conformational changes

Hemoglobin - two affinity states

induced by ligand binding
Molecular disease of Hemoglobin
 Sickle-Cell anemia
• Mutation of single amino acid Val instead of Glu at position 6 in beta chain
• Replacement of charged Glu for Val makes hydrophobic contact on the surface
• Hemoglobin aggregates
Another examples of proteins
 Fibrous Proteins:
From Structure to Function

Function Structure Example

Tough, rigid, Cross-linked -helixes -keratin

hard (nails, horns) Rigid linker (S—S)

Tensile strength, Cross-linked triple-helixes Collagen

non-stretching Flexible linker (Lys-HyLys)
(tendons, cartilage)

Soft, flexible Non-covalently held -sheets

non-stretchy van der Waals interaction Silk fibroin
(egg sac, nest, web)
Structure of -Keratin in Hair
Chemistry of Permanent Waving
 Structure of Collagen
• Collagen is an important constituent of
connective tissue: tendons, cartilage, bones,
cornea of the eye
• Each collagen chain is a long Gly- and Pro-rich
left-handed helix
• Three collagen chains intertwine into a right-
handed superhelical triple helix
• The triple helix has higher tensile strength than a
steel wire of equal cross section
• Many triple-helixes assemble into a collagen fibril
 4-Hydroxyproline in Collagen
• Forces the proline ring into a favorable folding
• Offer more hydrogen bonds between the three
strands of collagen
• The post-translational processing is catalyzed by
prolyl hydroxylase and requires -ketoglutarate,
molecular oxygen, and ascorbate (vitamin C)
 Silk Fibroin
• Fibroin is the main protein in silk from silk moths
and spiders
• Antiparallel  sheet structure
• Small side chains (Ala and Gly) allow the close
packing of sheets
• Structure is stabilized by
– hydrogen bonding within sheets
– London dispersion interactions between
sheets