Chapter 24

Proteins

 Introduction

The three major groups of biological polymers are

polysaccharides, proteins and nucleic acids
Proteins have many diverse functions; they are major components
of the following biomolecules
Enzymes and hormones which catalyze and regulate biological reactions
Muscles and tendons which provide the body with means for movement
Hemoglobin which carries oxygen to all parts of the body
Antibodies they are integral parts of the immune system

All proteins are polyamides

Their monomeric units are one of about 20 - amino acids

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 Proteins have several levels of structure

Primary structure refers to the exact sequence of amino acids along a protein

chain
Secondary and tertiary structures refer to the further bending and folding of the
primary structure
Quaternary structure refers to the aggregation of more than one polyamide chain

All amino acids except glycine are chiral and have the L
configuration (as related to glyceraldhyde) at the carbon

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 Amino acids
Structure and Names

22 amino acids but only 20 amino acids comprise the building

blocks for synthesis of proteins
The remaining 2 amino acids are derived by modification after
biosynthesis of the protein
Hydroxyproline and cystine are synthesized from proline and cysteine,

respectively, after the protein chain has been synthesized

Cysteine is oxidized under mild conditions to the dissulfide cystine

The reaction is reversible

This linkage is important in maintaining the overall shape of a protein

Essential Amino Acids

Essential amino acids are not made by higher animals and must be
part of the diet
There are 8 essential amino acids for adult humans (see Table 24.1)

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 Polypeptides and Proteins

Enzymes polymerize amino acids by forming amide linkages

The polymer is called a peptide and the amide linkages are called
peptide bonds or peptide linkages
Each amino acid in the peptide is called an amino acid residue
Proteins can contain one or more polypeptide chains and other
associated molecules or metal ions

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 Polypeptides are customarily written with the N-terminal residue to

the left
Three letter or one letter abbreviations are usually used as a short hand to

indicate the sequence of a polypeptide

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 Primary Structure of Polypeptides and Proteins

The sequence of amino acids in a polypeptide is called its primary

structure
Several methods exist to elucidate the primary structure of peptides

Edman Degradation

Edman degradation involve sequential cleavage and identification

of N-terminal amino acids
Edman degradation works well for polypeptide sequence analyses
up to approximately 60 amino acid residues
The N-terminal residue of the polypeptide reacts with phenyl isothiocyanate
The resulting phenylthiocarbamyl derivative is cleaved from the peptide chain

The unstable product rearranges to a stable phenylthiohydantoin (PTH) which is

purified by HPLC and identified by comparison with PTH standards

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 Examples of Polypeptide and Protein Primary

Structure

Oxytocin and Vasopressin

Oxytocin stimulates uterine contractions during childbirth

Vasopressin causes contraction of peripheral blood vessels and a
resultant increase in blood pressure
The two polypeptides are nonapeptides and differ in only 2 amino acid residues

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 Insulin

Insulin is a hormone which regulates glucose metabolism

Insulin deficiency in humans is the major cause of diabetes mellitus

The structure of bovine insulin (shown below) was determined in 1953 by Sanger
Human insulin differs from bovine insulin at only three amino acids in its

sequence

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 Secondary, Tertiary, and Quaternary Structures of

Proteins

Secondary Structure

The secondary structure of a protein is defined by local

conformations of its polypeptide backbone

These local conformations are specified in terms of regular folding patterns such

as helices, pleated sheets, and turns

The secondary structure of a protein is determined by the

sequence of amino acids in its primary structure
Key to secondary structure is that peptide bonds assume a
geometry in which all 6 atoms of the amide linkage are trans
coplanar

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 Coplanarity results from contribution of the second resonance

form of amides, in which there is considerable N-C double bond
character

The carbon with attached R groups between the amide nitrogen

and the carbonyl group has relatively free rotation and this leads
to different conformations of the overall chain

Two common secondary structure are the -pleated

sheet and the -helix

In the -pleated sheet, a polypeptide chain is in an extended

conformation with groups alternating from side to side

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 The extended polypeptide chains in -pleated sheets form

hydrogen bonds to adjacent polypeptide chains

Slight bond rotations are necessary between amide groups to avoid unfavorable

steric interactions between peptide side chains, leading to the pleated structure
The -pleated sheet is the predominant structure in silk fibroin

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 The -helix is the most important protein secondary structure

-Helices in a polypeptide are right-handed with 3.6 amino acid
residues per turn (See figure 24.11 page 1198)

The amide nitrogen has a hydrogen bond to an amino acid carbonyl oxygen that is

three residues away

The R groups extend away from the axis of the helix

-Helices comprise the predominant secondary structure of

fibrous proteins such as myosin (in muscle) and -keratin (in hair
and nails)
There are other secondary structures that are more difficult to
describe
Examples are coil or loop conformations and reverse turns or bends

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 Tertiary Structure

The tertiary structure of a protein is the three-dimensional shape

which results from further folding of its polypeptide chains
This folding is superimposed on the folding caused by its secondary structure

In globular proteins, the folding in tertiary structures exposes the

maximum number of polar (hydrophilic) side chains to the
aqueous environment, making most globular proteins water
soluble

The folding also serves to enclose a maximum number of nonpolar (hydrophobic)

side chains within the protein interior

Tertiary structures are stabilized by forces including hydrogen

bonding, disulfide bonds, van der Waals forces, and ionic
attractions

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 Quaternary Structure

The overall structure of a protein having multiple subunits is called

its quaternary structure
Not all proteins have quaternary structure

Hemoglobin

Hemoglobin is a globular protein that transports oxygen in the

blood
Hemoglobin contains four polypeptide subunits (2 designated ,
and 2 designated ) (See Figure 24.21, page 1210)

The subunits are shown in blue and green; subunits are shown in yellow and

cyan

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 Each of the four protein subunits carries a heme group

The four heme groups are shown in purple

Each heme group can bind one oxygen molecule in a reversible complex

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 Introduction

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are the

molecules that carry genetic information in the cell
DNA is the molecular archive for protein synthesis
RNA molecules transcribe and translate the information from DNA so it can be

used to direct protein synthesis

DNA is comprised of two polymer strands held together by

hydrogen bonds
Its overall structure is that of a twisted ladder

The sides of the ladder are alternating sugar and phosphate units
The rungs of the ladder are hydrogen-bonded pairs of heterocyclic amine bases

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 DNA polymers are very long molecules

DNA is supercoiled and bundled into 23 chromosomes for packaging in the cell

nucleus

The sequence of heterocyclic amine bases in DNA encodes the

genetic information required to synthesize proteins
Only four different bases are used for the code in DNA
A section of DNA that encodes for a specific protein is called a gene

The set of all genetic information coded by the DNA in an organism is its genome
The set of all proteins encoded in the genome of an organism and expressed at

any given time is its proteome

The sequence of the human genome is providing valuable

information related to human health

Example: A schematic map of genes on chromosome 19 that are related to

disease

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 Nucleotides and Nucleosides

Mild degradation of nucleic acids yields monomer units called

nucleotides
Further hydrolysis of a nucleotide yields:
A heterocyclic amine base

D-ribose (from RNA) or 2-deoxy-D-ribose (from DNA); both are C5 monosaccharides

A phosphate ion

The heterocylic base is bonded by a N-glycosidic linkage to C1 of

the monosaccharide
Examples: A general structure of an RNA nucleotide (a) and adenylic acid (b)

A nucleoside is a nucleotide without the phosphate group

A nucleoside of DNA contains 2-deoxy-D-ribose and one of the following four bases

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 A nucleoside of RNA contain the sugar D-ribose and one of the

four bases adenine, guanine, cytosine or uracil

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 Nucleosides that can be obtained from DNA

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 Nucleosides that can be obtained from RNA

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 Nucleotides can be named in several ways

Adenylic acid is usually called AMP (adenosine monophosphate)

It can also be called adenosine 5-monophosphate or 5-adenylic acid

Adenosine triphosphate (ATP) is an important energy storage

molecule
The molecule 3,5-cyclic adenylic acid (cyclic AMP) is an
important regulator of hormone activity

This molecule is biosynthesized from ATP by the enzyme adenylate cyclase

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 Deoxyribonucleic Acid: DNA

Primary Structure

The monomer units of nucleic acids are nucleotides

Nucleotides are connected by phosphate ester linkages

The backbone of nucleic acids consists of alternating phosphate

and sugar units
Heterocyclic bases are bonded to the backbone at each sugar unit
The base sequence contains the encoded genetic information
The base sequence is always specified from the 5 end of the
nucleic acid

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 Secondary Structure

The secondary structure of DNA was proposed by Watson and

Crick in 1953
E. Chargaff noted that in DNA the percentage of pyrimidine bases
was approximately equal to the percentage of purine bases
Also the mole percentage of adenine Is nearly equal to that of thymine
The mole percentage of guanine is nearly equal to cytosine

Chargaff also noted that the ratio of A and T versus G and C varies
by species but the ratio is the same for different tissues in the
same organism

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 X-ray crystallographic data showed the bond lengths and angles of

purine and pyrimidine bases
X-ray data also showed DNA had a long repeat distance (34 )

Based on this data, Watson and Crick proposed the double helix
model of DNA (next slide)
Two nucleic acid chains are held together by hydrogen bonding between the

bases on opposite strands

The double chain is wound into a helix
Each turn in the helix is 34 long and involves 10 successive nucleotide pairs
Each base pair must involve a purine and a pyrimidine to achieve the proper
distance between the sugar-phosphate backbones
Base pairing can occur only between thymine and adenine, or cytosine and
guanine; no other pairing has the optimum pattern of hydrogen bonding or would
allow the distance between sugar-phosphate backbones to be regular

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 Specific pairing of bases means the two chains of DNA are

complementary

Knowing the sequence of one chain allows one to also know the sequence of the

other

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 Replication of DNA (see next slide)

The DNA strand begins to unwind just prior to cell division

Complementary strands are formed along each chain (each chain
acts as a template for a new chain)
Two new DNA molecules result; one strand goes to each daughter
cell

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 RNA and Protein Synthesis

The central dogma of molecular genetics

A gene is the portion of a DNA molecule which codes for one

protein

Proteins have many critical functions, e.g., catalysis, structure, motion, cell

signaling, the immune response, etc.

DNA resides in the nucleus and protein synthesis occurs in the

cytoplasm
Transcription of DNA into messenger RNA (mRNA) occurs in the nucleus

mRNA moves to the cytoplasm and the translation into proteins occurs using two

other forms of RNA: ribosomal RNA (rRNA) and transfer RNA (tRNA)

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 Transcription: Synthesis of Messenger RNA (mRNA)

In the nucleus a DNA molecule partially unwinds to expose a

portion corresponding to at least one gene
Ribonucleotides with complementary bases assemble along the
DNA strand
Base-pairing is the same in RNA, except that in RNA uracil replaces thymine

Ribonucleotides are joined into a chain of mRNA by the enzyme

RNA polymerase

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 An intron (intervening sequence) is a segment of DNA which is

transcribed into mRNA but not actually used when a protein is
expressed
An exon (expressed sequence) in the part of the DNA gene which
is expressed
Each gene usually contains a number of introns and exons
Introns are excised from mRNA after transcription

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 Ribosomes - rRNA

Protein synthesis is catalyzed in the cytoplasm by ribosomes

A ribosome consists of approximately two thirds RNA and one third protein
A ribosome is a ribozyme ( an reaction catalyst made of ribonucleic acid)

A ribosome has 2 large subunits

The 30S subunit binds the mRNA that codes for the protein to be translated
The 50S subunit catalyzes formation of the amide bond in protein synthesis

Transfer of an amino acid to the growing peptide chain is aided by

acid-base catalysis involving an adenine in the 50S subunits

See Figure 25.14, page 1238

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 Transfer RNA (tRNA)

Transfer RNAs (tRNAs), specific to each amino acid, transport

amino acids to complimentary binding sites on the mRNA bound
to the ribosome
More than one tRNA codes for each amino acid

tRNA is comprised of a relatively small number of nucleotides

whose chain is folded into a structure with several loops
One arm of the tRNA always terminates in the sequence cytosine-cytosine-

adenine, and it is here the amino acid is attached

On another arm is a sequence of three bases called the anticodon, which binds
with the complementary codon on mRNA

The mRNA genetic code is shown on the next page

The structure of a tRNA molecule is shown in Figure 25.15, page
1240

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 The Genetic Code

The genetic code is based on three-base sequences in mRNA

Each three-base sequence corresponds to a particular amino acid
The fact that three bases are used to code for each amino acid provides

redundancy in the overall code and in the start and stop signals
N-formyl methionine (fMet) is the first amino acid incorporated into bacterial
protein and appears to be the start signal
fMet is removed from the protein chain before its synthesis is complete

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 Translation

Translation is peptide synthesis by a ribosome using the code

from an mRNA
The following is an example (see figure on next page):
An mRNA binds to a ribosome

A tRNA with the anticodon for fMet associates with the fMet codon on the mRNA
A tRNA with anticodon UUU brings a lysine residue to the AAA mRNA codon
The 50S ribosome catalyzes amide bond formation between the fMET and lysine
The ribosome moves down the mRNA chain to the next codon (GUA)
A tRNA with the anticodon CAU brings a valine residue
The ribosome catalyzes amide bond formation between Lys and Val
The ribosome moves along the mRNA chain and the process continues, e.g., with

the tRNA for phenylalanine binding to the ribosome

A stop signal is reached and the ribosome separates from the mRNA
At this point the polypeptide also separates from the ribosome

The polypeptide begins to acquire its secondary and tertiary

structure as it is being synthesized
Several ribosomes can be translating the same mRNA molecule
simultaneously
Protein molecules are synthesized only when they are needed

Regulator molecules determine when and if a particular protein will be expressed

i.e. synthesized

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 Determining the Base Sequence of DNA

The Chain-Terminating (Dideoxynucleotide) Method

DNA molecules are replicated in such a way that a family of partial copies is

generated; each DNA copy differs in length by only one base

Random chain-termination is done by poisoning a replication reaction with a low
concentration of 23-dideoxynucleotides, which are incapable of chain elongation at
their 3 position
The 23-dideoxynucleotides are labeled with covalently attached colored fluorescent
dye molecules, with each color representing a base type

The partial copies are separated according to length by capillary electrophoresis

The terminal base on each strand is detected by the color of laser-induced

fluorescence as each DNA molecule passes the detector

A four-color chromatogram is generated (see Figure 25.17, page 1246)

Automation of high-throughput dideoxy sequencing made possible

completion of the Human Genome Project by the 50th anniversary of
Watson and Cricks elucidation of the structure of DNA in 2003

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 The Polymerase Chain Reaction (PCR)

PCR is an extraordinarily simple and effective method for

exponentially multiplying (amplifying) the number of copies of a
DNA molecule.
PCR beginning with a single molecule can lead to 100 billion copies in an

afternoon
The Nobel Prize was awarded to K. Mullis in 1993 for invention of PCR

PCR requires:

A sample of the DNA to be copied

The enzyme DNA polymerase
A short primer sequence complimentary to the template DNA
A supply of A, C, G, and T nucleotide triphosphate monomers
A simple device for thermal cycling during the reaction sequence

The PCR process is summarized on the next 2 slides

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