Chapter 4:
Protein Structure and
Folding
Life … is a relationship between
molecules.
Linus Pauling, as quoted in T. Hager, Force of
Nature: The Life of Linus Pauling (1997), p. 542
4.1 Introduction
• Proteins are found in all living systems, ranging
from bacteria and archaea through the
unicellular eukaryotes, to plants, fungi, and
animals.
• In all life forms, proteins are made up of the
same building blocks―amino acids.
• Each cell contains thousands of different genes
and makes thousands of different proteins.
What is a gene?
• In the late 1930s…
“A molecule of living stuff made up of
many atoms held together.”
What is a gene?
• A specific stretch of nucleotides in DNA (or in
some viruses, RNA) that contains information
for making a particular RNA molecule that in
most cases is used to make a particular protein.
4.2 Primary structure: amino
acids and the genetic code
The 22 amino acids found in
proteins
• Proteins are chain-like polymers of amino acids
specified by the genetic code.
• Each amino acid has an amino group (NH3+)
and a carboxyl group (COO) attached to a
central carbon called the -carbon.
• The only difference between two amino acids
is in their different side chain or “R group.”
• At pH 7 the amino and carboxyl groups of
amino acids are charged.
• Over a pH range from 1 to 14 these groups
exhibit binding and dissociation of a proton.
• The weak acid-base behavior of amino acids
provides the basis for many techniques for
amino acid identification and protein
separations.
Protein primary structure
• Amino acids joined together by peptide
bonds form the primary structure of a
protein.
• The amino group of one molecule reacts
with the carboxyl group of the other in a
condensation reaction.
• When joined in a series of peptide bonds,
amino acids are called residues.
• A short sequence of amino acids is called a
peptide; the term polypeptide applies to longer
chains of amino acids.
• The arrangement of amino acids, with their
distinct side chains, gives each protein its
characteristic structure and function.
• The peptide bond has a partial double bond
character as a result of resonance.
• Free rotation occurs only between the -carbon
and the peptide unit.
• Trans and cis-configurations are possible about
the rigid peptide bond.
• The peptide chain is flexible, but it is more rigid
than it would be if there were free rotation about
all of the bonds.
Translating the genetic code
• How is the genetic code translated into a
specific sequence of amino acids?
• The mechanism of translation is
described in detail in Chapter 14.
• A DNA sequence is read in triplets using the
antisense (non-coding) strand as a template
that directs synthesis of RNA via
complementary base pairing.
• An open reading frame (ORF) in the mRNA
indicates the presence of a start codon
followed by codons for a series of amino acids
and ending with a termination codon.
The genetic code
• Each “codon box” is composed of four threeletter codes, 64 in all.
• 61 codons are recognized by tRNAs for the
incorporation of the 20 common amino acids.
• 3 codons signal termination, or code for
selenocysteine and pyrrolysine.
The genetic code is degenerate
• tRNAs specific to a particular amino acid
recognize multiple codon triplets that
differ only in the third letter.
e.g. leucine is coded for by 6 different codons,
while methionine has only one codon
The “wobble hypothesis”
• Pairing between codon and anticodon at
the first two codon positions always
follows the usual rule of complementary
base pairing.
• Exceptional “wobbles” (non-Watson-Crick
base pairing) can occur at the third
position.
The genetic code is not universal
• In certain organisms and organelles the
meaning of select codons has been
changed.
e.g. Tetrahymena reads UAA and UAG as
glutamine (Gln)
The 21st and 22nd genetically
encoded amino acids
The UGA code for selenocysteine is found in:
• >15 genes in prokaryotes that are involved in
redox reactions.
• >40 genes in eukaryotes that code for various
antioxidants and the type I iodothyronine
deiodinase.
The UAG code for pyrrolysine has been found in:
• a few archaebacteria and eubacteria.
Modified nucleotides and codon bias
• “Wobbles” can occur at the third position.
• When bases in the anticodon are modified,
further pairing patterns are possible.
• Examples:
Inosine can pair with U, C, and A.
2-thiouracil restricts pairing to A alone.
Implications of codon bias for
molecular biologists
• The frequencies with which different codons are used
vary significantly between different organisms and
between proteins expressed at high or low levels within
the same organism.
• Expression of functional proteins in heterologous hosts
is a cornerstone of molecular biology research.
• Codon bias can have a major impact on the efficiency
of expression of proteins if they contain codons that are
rarely used in the desired host.
• What might happen if you tried to
express a Tetrahymena gene that
encodes a glutamine-rich protein in E.
coli?
D-
and L-amino acids in nature
• D- and L-amino acids are enantiomers
(sterioisomers that are mirror images of
each other).
• Living organisms are composed
predominantly of L-amino acids.
• Ribosomes only use L-amino acids to
make proteins.
Exceptions:
• D-amino acids are found in some peptides
in microorganisms, but are synthesized by
pathways that do not involve the ribosome.
• D-amino acids are present in some
peptides in other organisms, but are made
from the genetically encoded L-amino
acids by a post-translational process.
Examples:
• D-amino acids are present in the venom of
some bivalves, snails, spiders,
amphibians, and the duck-bill platypus.
• The presence of D-amino acids is linked to
more potent venom.
4.3 The three-dimensional
structure of proteins
• There is tremendous variation in the size
and complexity of proteins.
• Dalton (Da) units are typically used to
describe the molecular weight of proteins.
• Typical polypeptide chains have
molecular weights of 20 to 70 kDa
(20,000 to 70,000 Da).
• The average molecular weight of an
amino acid is 110.
• A typical polypeptide chain thus contains
181 to 636 amino acids.
Secondary structure
• Interactions of amino acids with their neighbors
gives a protein its secondary structure.
• Primarily stabilized by hydrogen bonds.
• Also depends on disulfide bridges, van der
Waals interactions, hydrophobic contacts, and
electrostatic interactions.
The three basic elements of protein
secondary structure
• -helix
• -pleated sheet
• Unstructured turns
-helix
• Most common structural motif in proteins.
• Tight helical structure stabilized by
hydrogen bonding among near-neighbor
amino acids.
• Proline, the “helix-breaking residue”,
cannot participate as a donor in hydrogen
bonding.
-pleated sheet
• Extended amino acids chains packed
side by side to create a pleated,
accordian-like appearance.
• Stabilized by hydrogen bonding.
Parallel  structure
• Two segments of a polypeptide chain (or two
individual polypeptides) are aligned in the Nterminal to C-terminal direction or vice versa.
Antiparallel  structure
• One segment is N-terminal to C-terminal and
the other is C-terminal to N-terminal.
Unstructured turns
• “Turns” connect the -helices and pleated sheets in proteins.
• Relatively short loops that do not exhibit
a defined secondary structure.
Tertiary structure
• The folded three-dimensional shape of a
polypeptide.
• Most interactions are stabilized by
noncovalent bonds:
Hydrophobic interactions
Hydrogen bonds
• The principle covalent bonds within and
between polypeptides are disulfide (S-S)
bonds or “bridges” between cysteines.
Three main categories of tertiary
structure
• Globular proteins
• Fibrous proteins
• Membrane proteins
Globular proteins
• The overall shape of most proteins is
roughly spherical.
e.g. the enzyme lysozyme folds up into a
globular tertiary structure forming the active
site.
Fibrous proteins
• Long filamentous or “rod-like” structures.
• Structural components of cells and
tissues.
• A number of major designs:
- triple helical arrangement
- “coiled coils”
- antiparallel -pleated sheets
Membrane proteins
• Differ from soluble proteins in the relative
distribution of hydrophobic amino acid
residues.
• The seven transmembrane helix
structure is a common motif in membrane
proteins.
Prediction of protein structure
• By comparing the sequences of proteins
of unknown structure with those that
have been determined, it is often
possible to make structural predictions
based on identified similarity.
Quaternary structure
• A functional protein can be composed of
one or more polypeptide subunits.
• Can be identical or nonidentical subunits.
• Stabilizing bonds are the same as those
for tertiary structure.
• Quaternary structure allows greater
versatility of function.
• Catalytic or binding sites are often
formed at the interface between subunits.
e.g. the two  and two  subunits in
hemoglobin form a binding site for a heme
group
4.4 Protein function and
regulation of activity
• Proteins larger than about 20 kDa are often
formed from two or more domains with specific
functions.
• A single domain is usually formed from a
continuous amino acid sequence.
e.g. DNA-binding domain
• Domains can contain common structuralfunctional motifs.
• Proteins have a diversity of functions in
cells.
• One vital role of proteins is to serve as
enzymes that catalyze the hundreds of
chemical reactions necessary for life.
Enzymes are biological catalysts
• Enzymes lower the activation energies of the
chemical groups that participate in a reaction
and thereby speed up the reaction.
• The substrate forms a tight complex with the
enzyme by binding to a region called the active
site.
• Most enzymes act through an induced-fit
mechanism.
Example:
• Lysozyme catalyzes the breakdown of
polysaccharides from the E. coli peptidoglycan
layer.
• The active site is a long, deep cleft that can bind
six N-acetylglucosamine (NAG) and Nacetylmuramic acid (NAM) units.
• Lysozyme brings the reacting species together
in a geometry that favors reaction.
• For the fourth NAG-NAM unit to fit in the
active site, it must be distorted, and forms
a less stable conformation.
• Asp 52 and Glu35 residues of lysozyme
interact with the fourth and fifth NAG-NAM
units, breaking the C-O bond between
them by hydrolysis.
Regulation of protein activity by
post-translational modifications
The functional activity of proteins can be
regulated at several different levels:
•Transcription
•RNA processing
•Translation
•Post-translational modifications, such as
phosphorylation and allosteric effectors
• After translation, proteins are joined
covalently and noncovalently to other
molecules.
e.g. lipoproteins, glycoproteins,
metalloproteins
• The most common regulatory mechanism
is the reversible phosphorylation of
amino acid side chains.
Protein phosphorylation
• May cause a protein to change shape and
unmask or mask a catalytic or functional
domain.
• Phosphorylated side chain may be part of a
binding motif to facilitate formation of a
multiprotein complex.
• Phosphorylated side chain may promote
dissociation of a multiprotein complex.
Kinases
• Catalyze the addition of phosphate groups.
• Tend to be very specific, acting on very few
substrates.
• Two protein kinase groups have been widely
studied in eukaryotes:
1. Those that phosphorylate serine or threonine side
chains.
2. Those that phosphorylate tyrosine side chains.
Phosphatases
• Remove phosphates.
• Tend to be less specific, acting on many
substrates.
Allosteric regulation of
protein activity
• Ligand-induced conformational change.
• An active site or another binding site is
altered in a way that increases or
decreases its activity.
Example:
• Cyclin-dependent kinase (CDK) activity is
regulated by both allosteric modification
and phosphorylation.
Inactive conformation of CDK
• The T loop is located at the entrance to the
active site.
• Polypeptide substrates are blocked from
gaining access to the ATP molecule in the
active site.
• A critical glutamate residue in the PSTAIRE
helix is held at a distance from the active site.
Partial activation of CDK
• Binding of cyclin to CDK induces a
conformational change.
• T loop moves away from the entrance of the
active site.
• Critical glutamate in PSTAIRE helix moves
into active site.
Full activation of CDK
• Phosphorylation of Thr160 in T loop by
CDK-activating kinase (CAK).
• Stabilizes active site “catalytic cleft.”
Macromolecular assemblages
• Expression of the genetic information
relies on the sequential action of large
and dynamic macromolecular
assemblages or “molecular machines.”
4.5 Protein folding and
misfolding
• In some cases, protein folding is initiated
before the completion of protein
synthesis.
• Other proteins undergo major folding
after release into the cytoplasm or a
specific organelle.
• Most proteins require “molecular
chaperones” to fold properly in vivo.
Molecular chaperones
• Increase the efficiency of protein folding.
• Reduce the probability of competing reactions
such as aggregation.
• Aid in the destruction of misfolded proteins.
• Typically ATP-dependent.
• Heat-shock proteins promote protein folding
and aid in the destruction of misfolded protein.
e.g. Hsp40, Hsp70, Hsp90
• Hsp90 mediates protein folding by undergoing
major shape changes upon binding and
hydrolysis of ATP and interaction with p23.
Endoplasmic reticulum “quality control”
• Secreted proteins are translocated into the
endoplasmic reticulum (ER).
• Folding takes place before secretion through
the Golgi apparatus.
• Folding catalysts accelerate potentially slow
steps in the folding process
e.g. peptidylprolyl and protein disulfide isomerases
• Incorrectly folded proteins are detected by the
“unfolded protein response” and targeted for
degradation.
Ubiquitin-mediated
protein degradation
• Ubiquitin (a 76 amino acid polypeptide) is
attached to a protein by a series of enzymemediated reactions.
• The ubiquitin-conjugated protein is then
targeted to the 26S proteasome.
• Ubiquitin is released and the target protein is
degraded by proteases.
Protein misfolding diseases
• Formation of protein aggregates is linked to at
least 20 different human diseases.
• Normally soluble proteins accumulate as
insoluble deposits known as amyloid or
amyloid-like fibrils.
• Proteins in amyloid-like fibrils fold into a cross
-spine.
Prions
The primary cause of transmissible
spongiform encephalopathies (TSEs).
•Progressive neurodegeneration.
•Dementia.
•Loss of muscle control of voluntary movements.
•Once symptoms appear, death results in 6
months to 1 year.
•There is no cure.
Human forms of prion disease
•
•
•
•
Kuru
Creutzfeldt-Jakob disease
Gerstmann-Straussler syndrome
Fatal familial insomnia
Animal forms
• Scrapie (sheep)
• Bovine spongiform encephalopathy (BSE: “mad cow
disease”)
• Chronic wasting disease (elk and deer)
The “prion only” hypothesis
of infection
• Stanley Prusiner: Nobel Prize in 1997.
• Lack of immune response characteristic of
infectious diseases.
• Long incubation time (up to 40 years for kuru).
• Resistance of the infectious agent to radiation
that destroys living microorganisms (e.g.
viruses, bacteria).
• The infectious agent is not a living
organism but a protein called PrPSc with
the unusual ability to replicate itself within
the body.
• The prion PrPSc has the same amino acid
sequence as the normal host protein
PrPC.
• But, the prion is misfolded into a different
3-D structure.
After misfolding the prion protein
becomes…
• Aggregated (brain plaques).
• Protease resistant.
• Infectious.
• Able to survive standard sterilization
techniques.
Normal cell
• The normal cellular protein PrPC is a cell
surface protein expressed in neurons.
Infected cell
• Host protein PrPC is misfolded to form new
prions called PrPSc.
• Formation of fibrils, aggregates, and amyloid
plaques.
Human sporadic transmissible
spongiform encephalopathies
• PrPC misfolds spontaneously and generates
more prions by “autoinfection”
Creutzfeldt-Jakob disease (CJD)
• Preventative action?
None – frequency of one in a million.
Human inherited transmissible
spongiform encephalopathies
• Mutated PrPC gene with greater tendency to
spontaneously misfold to prion form.
Gerstmann-Straussler syndrome
Fatal familial insomnia
• Preventative action?
None – 100% likelihood of disease progression.
Human infectious transmissible
spongiform encephalopathies
• Eating brains or infected meat products
Kuru: former ritualistic cannibalism in Papua New
Guinea
New variant CJD: consumption of tainted beef
• Preventative action?
Don’t eat contaminated meat products.
Pathway from infection to disease
• Penetration
• Translocation
• Multiplication
• Pathogenesis