Fundamental

Molecular Biology
Second Edition

Lisabeth A. Allison

Chapter 4
Protein Structure and Folding

Copyright © 2012 John Wiley & Sons, Inc. All rights reserved.
Cover photo: Julie Newdoll/www.brushwithscience.com “Dawn1 of the
Double Helix”, oil and mixed media on canvas, © 2003
 Outline
4.1 Introduction
4.2 Primary structure: amino acids and the genetic code
4.3 The three-dimensional structure of proteins
4.4 Protein function and regulation of activity
4.5 Protein folding and misfolding

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 4.1

• 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.

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 What is a gene?

• In the late 1930s…

“A molecule of living stuff made up of many atoms
held together.”

• 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.

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 4.2

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.”

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 pyrrolysine

selenocysteine

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• 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 (Fig. 4.2).

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 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.

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• 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.

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• The peptide bond has a partial double bond character
as a result of resonance (Fig. 4.4A).

• Free rotation occurs only between the -carbon and the

peptide unit.

• Trans and cis-configurations are possible about the

rigid peptide bond (Fig. 4.4B).

• The peptide chain is flexible, but it is more rigid than it

would be if there were free rotation about all of the
bonds.
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 Translating the genetic code

• 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 (Fig. 4.5).

• 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.

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15
 The genetic code
• Each “codon box” is composed of four three-letter
codes, 64 in all (4 x 4 x 4).

• 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.

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 *
*

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 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

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 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.
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 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)

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21
 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.

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 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.

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 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.
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• What might happen if you tried to express a
Tetrahymena gene that encodes a glutamine-rich
protein in E. coli?

• (Tetrahymena reads UAA and UAG as glutamine (Gln)

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 D- and L-amino acids in nature

• D- and L-amino acids are enantiomers (stereoisomers

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.

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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.

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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.

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(from spider venom)

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 4.3

• 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

Da.

• A typical polypeptide chain thus contains 181 to 636

amino acids.

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 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.

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 The three basic elements of protein
secondary structure

 -helix

 -pleated sheet

• Unstructured turns

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34
 -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.

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 -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 N-terminal 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.
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 Unstructured turns

• “Turns” connect the -helices and -pleated sheets

in proteins.

• Relatively short loops that do not exhibit a defined

secondary structure.

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 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 (Fig. 4.9).

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39
Three main categories of tertiary structure

• Globular proteins

• Fibrous proteins

• Membrane proteins

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 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.

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 Fibrous proteins
• Long filamentous or “rod-like” structures.

• Structural components of cells and tissues.

• A number of major designs:

- triple helical arrangement (e.g. collagen)

- “coiled coils”
- antiparallel -pleated sheets

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 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.

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 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.

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• 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

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 4.4

• 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 contain common structural-functional 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.
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 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.

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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 N-acetylmuramic
acid (NAM) units.

• Lysozyme brings the reacting species together in a

geometry that favors reaction.

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• For the fourth NAG-NAM unit to fit in the active
site, it must be distorted, and forms a less stable
conformation.

• Asp52 and Glu35 residues of lysozyme interact

with the fourth and fifth NAG-NAM units,
breaking the C-O bond between them by
hydrolysis.

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52
 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

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54
• 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.

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 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.

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 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.

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 Phosphatases

• Remove phosphates.

• Tend to be less specific, acting on many substrates.

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 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.

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Negative control Positive control

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Example:

• Cyclin-dependent kinase (CDK) activity is regulated

by both allosteric modification and phosphorylation.

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Inactive conformation of CDK (Fig. 4.19)

• 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.

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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.

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Full activation of CDK

• Phosphorylation of Thr160 in T loop by CDK-

activating kinase (CAK).

• Stabilizes active site “catalytic cleft.”

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(CDK)

Phosphorylation of Thr160

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 Macromolecular assemblages
• Expression of the genetic information relies on the
sequential action of large and dynamic macromolecular
assemblages or “molecular machines.”

• 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.
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67
 4.5
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.

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Regulation of protein folding
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• 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 (Fig. 4.21).

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Hsp90 chaperone function

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 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. 72
 Ubiquitin-mediated protein degradation

• Ubiquitin (a 76 amino acid polypeptide) is attached to a

protein by a series of enzyme-mediated reactions.

• The ubiquitin-conjugated protein is then targeted to the

26S proteasome.

• Ubiquitin is released and the target protein is degraded

by proteases.

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Ubiquitin Proteasome System programme

https://www.youtube.com/watch?v=hvNJ3yWZQbE

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 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.

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Amyloid-like fibrils

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 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

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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)

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 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).

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• The infectious agent is not a living organism but a
protein called scrappie prion protein (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.

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After misfolding the prion protein becomes…

• Aggregated (brain plaques)

• Protease resistant

• Infectious

• Able to survive standard sterilization techniques

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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.

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 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.

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 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.

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 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.

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 Outline
4.1 Introduction
4.2 Primary structure: amino acids and the genetic code
4.3 The three-dimensional structure of proteins
4.4 Protein function and regulation of activity
4.5 Protein folding and misfolding

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