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Topic 2

PROTEINS PROTEINS
 Amino acid
Structure and Function  Peptide bonds
 Levels of protein structure

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What are PROTEINS?
 Proteins are macromolecules.
 They are constructed from one or
more unbranched chains of amino
acids; that is, they are polymers.
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Cellular Functions of Proteins
• Are biological catalysts (enzymes)
• Are antibodies that fight antigens (bacteria
and viruses)
• Transport molecules and ions
• Regulate cell function
• Provide structural support and mechanical
strength
• Are necessary for all forms of movement
• Are sources of amino acids for growth
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The α-Amino Acids Stereochemistry of the α-Amino Acids

Building blocks of
protein

An alpha amino
acid is a carboxylic
acid with an amino
group on the
carbon alpha to
the carboxylic
the standard (DNA-encoded) amino but…D-amino acids are occasionally
acid. acids found in proteins possess the L produced by certain organisms, but
stereochemistry are not found in their proteins

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The 20 Standard Amino Acids
20 amino acids form amides in proteins
All are -amino acids - the amino and carboxyl are
connected to the same C
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The 20 Standard Amino Acids
They differ by the other substituent attached to the
 carbon, called the side chain, with H as the
fourth substituent except for proline
Proline, is a five-membered secondary amine,
with N and the  C part of a five-membered ring
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Protein-derived amino acids

1. All 20 are α-amino acids.

2. For 19 of the 20, the α-amino group is
primary; for proline, it is secondary.
3. With the exception of glycine, the α-
carbon of each is a stereocenter.
4. Isoleucine and threonine each contain
a second stereocenter.

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Essential and Nonessential Amino Acids Zwitterions

In neutral solution, the COOH is ionized and the NH2 is
protonated

The resulting structures have “+” and “-” charges (a

dipolar ion, or zwitterion)

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Ionization vs. pH Ionization vs. pH

The net charge on an amino acid depends on If we add a strong base such as NaOH to the solution
and bring its pH to 10.0 or higher, a proton is
the pH of the solution in which it is dissolved.
transferred from the NH3+ group to the base turning the
zwitterion into a negative ion.
If we dissolve an amino acid in water, it is present in
the aqueous solute as its zwitterion. O O
+ - - -
If we now add a strong acid such as HCl to bring the H3 N-CH-C-O + OH H2 N-CH-C-O + H2 O
pH of the solution to 2.0 or lower, the strong acid R R
donates a proton to the -COO- of the amino acid to summarize:
turning the zwitterion into a positive ion. O O - O
+ OH- + - OH
H3 N-CH-C-OH H3 N-CH-C-O H2 N-CH-C-O-
O O +
H3 O+
+ - +
+ R H3 O R R
H3 N-CH-C-O + H3 O H3 N-CH-C-OH + H2 O pH 2.0 pH 5.0 - 6.0 pH 10.0
R R Net charge +1 Net charge 0 N et ch arge -1

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Ionization vs. pH Isoelectric point (pI)

Isoelectric
to summarize:
point, pI:
The pH at
O O - O which the
+ OH- + - OH
H3 N-CH-C-OH H3 N-CH-C-O H2 N-CH-C-O-
+
H3 O H3 O+ majority of
R R R
pH 2.0 pH 5.0 - 6.0 pH 10.0 molecules of a
Net charge +1 Net charge 0 N et ch arge -1 compound in
solution have
no net charge.

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Titration of amino acids

When an amino
acid is titrated,
the titration curve
represents the
reaction of each
functional group
with the
hydroxide ion

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Titration of alanine with NaOH

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Titration of histidine with NaOH Abbreviations and Codes

Alanine A, Ala Leucine L, Leu
Arginine R, Arg Lysine K, Lys
Asparagine N, Asn Methionine M, Met
Aspartic acid D, Asp Phenylalanine F, Phe
Cysteine C, Cys Proline P, Pro
Glutamine Q, Gln Serine S, Ser
Glutamic Acid E, Glu Threonine T, Thr
Glycine G, Gly Tryptophan W, Trp
Histidine H, His Tyrosine Y, Tyr
Isoleucine I, Ile Valine V, Val

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Learning the Names and Codes UV Spectra of the Aromatic Amino Acids
The names are not systematic so you learn them by
using them (They become your friends)
Tryptophan and
• If only one amino acid begins with that letter, use it
(Cys, His, Ile, Met, Ser, Val) tyrosine account
• If more than one begins with that letter, the more for most of the UV
common one uses the letter (Ala, Gly, Leu, Pro, Thr) absorbance by
• For the others, some are phonetic: Fenylalanine, proteins in the
aRginine, tYrosine region around 280
• Trp has a double ring, hence W nm.
• Amides have letters from the middle of the alphabet
(Q – Think of “Qtamine” for glutamine; asparagine -
contains N
• “Acid” ends in D and E follows (smallest is first:
aspartic aciD, Glutamic acid E)

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Cysteine and disulfide formation Nonstandard and Modified Amino Acids

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Peptides and the Peptide Bond

Amino acids can be

covalently linked by
formation of an amide
bond between α-amino
and α-carboxyl group.

The bond is called a

peptide bond.

The linkage produces

a peptide.

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CIS CONFIGURATION TRANS CONFIGURATION

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Polypeptide structure & trans amide bonds
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Peptides: Structure and Names
Structure left (N-term) to right (C-term)
Structure is based on the repeating sequence
N-C-C-N-C-C-N-C-C
N is the a-amino group; red is the α-carbon; and
blue is the carbonyl carbon.
Name peptides by prefixing (L to R) the amino acid
name , the ending –ine changed to –yl, and terminating
in the COOH end AA name.
Example: ala-gly is alanylglycine
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Polypeptide Structure Practice

Write the structure of the ff. peptides at pH 7

a) threonylcysteine
b) Isoleucylmethionylaspartate
c) tyrosylglycylglycylphenylalanylmethionine
d) glycylvalylserine
e) phenylalanyltyrosylleucine

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Side Chain Ionization of Peptides
Rule:
if the solution pH < pKa of
the functional group, the
group is protonated;
if the solution pH > pKa of
the functional group, the
group is unprotonated
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Biologically Important Peptides
Glutathione (GSH)
• protects cells from the destructive effects of oxidation
by reacting with substances such as peroxides.
• in red blood cells: glutathione protects against
formation of methemoglobin by reducing H2O2 in a
reaction catalyzed by the enzyme glutathione
peroxidase
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Biologically Important Peptides The Hierarchy of Protein Structure

Met-enkephalin Leu-enkephalin
• small peptide hormones synthesized in the brain
• neuropeptide hormones
Primary – amino acid sequence
• inhibit motility and blood flow to the gastrointestinal Secondary - local structures
tract Tertiary -overall 3-dimensional shape
• play a role in pain perception, perhaps serving as a Quaternary - subunit organization
pain blockage

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Primary Structure Importance of the Primary Structure

 The primary structure Sickle –cell anemia
of a protein is the
amino acid sequence - sickle-cell anemia results from a single
of the chain. mutation in the normal Hb protein (Hb A)
 Results from the
covalent bonding - Glu→Val at position 6 in the "β-chain” results in
between the amino Hb S
acids in the chain.
- Val creates a hydrophobic'sticky' patch on the
 Primary structures are Hb surface that causes the proteins to
translations of associate abnormally and form long,fibrous
information contained aggregates
in the genes.
- the aggregates give erythrocytes a 'sickled’
appearance, and weakens them as well as
increasing capillary blockage

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Sickle-cell anemia Sickle-cell anemia

Normal erythrocytes Sickle-cell erythrocytes

the aggregates give erythrocytes a 'sickled’
appearance, and weakens them as well as
increasing capillary blockage

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Secondary Structure The α-helix

A rigid, rodlike
The two very important secondary structures of structure that forms
proteins are: when a polypeptide
α-helix chain twists into a
right-handed helical
β-pleated sheet conformation.

Both depend on hydrogen bonding between the Every carbonyl oxygen

amide H and the carbonyl O further down the H-bonded to an amide
chain or on a parallel chain. hydrogen four amino
acids away in the
chain.

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The α-helix The α-helix

The repeat distance

of the helix, or its
pitch, is 5.4 Å, and
there are 3.6 amino
acids per turn of the
helix

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Hydrogen bonding in the α-helix Hydrogen bonding in the α-helix

Every backbone
amide group is engaged in The internal H-bonds
H-bonding as both donor are between the i and
(carbonyl O) and acceptor i+3 residues, and all
(amide N-H, except for carbonyls 'point' in the
the first four nitrogens at same direction
the N-terminus and the
first four carbonyls at the
C-terminus

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The α-helix and the R group Factors that stabilize the α-helix
electrostatic bonding interactions
amino acid side chains are between Arg103

projected towards outside (a) adjacent residues or

(b) residues 3 or 4 residues apart
(as counting along backbone)

large R-groups or repulsive Asp100

electrostatic charges between
adjacent residues can be
destabilizing

α-helices do not like to have Pro

or Gly residues
(i.e., they are destabilizing)

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The α-helix has an overall dipole The β sheet

also called β-pleated sheets
negatively charged peptide backbone is in an extended conformation (β-strand )
amino acids often
found here (stabilize
the positive charge
Intramolecular
of the dipole) H-bonds adjacent
β-strands
run in
opposite
directions
H-bonds between
positively charged adjacent β-strands
amino acids often are 'parallel’
found here (stabilize Side chain groups
the negative charge alternate between
of the dipole) projecting above
& below

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The β sheet Some proteins contain both α-helices & β-sheets

• α -helices can gradually

curve, and are not exactly
straight, and that in the β -
adjacent sheet the strands are not the
β-strands same in length, nor is each
run in
same perfectly 'flat’
H-bonds between directions
adjacent β-strands
are ‘angled’ • some parts that do not
have either α-helical or
β-sheet character--these
Side chain groups
alternate between regions may be 'disordered',
projecting above or may represent a type of
& below
turn structure

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Fibrous Proteins and Secondary Structure The α-keratins

Fibrous proteins are distinguished from globular
A fibrous structural
proteins by their filamentous or elongated form. protein consists of
~300 residues coiled
Play a structural roles in animal cells and tissues. into a right-handed
helical secondary
structure, -helix
stabilized by H-bonds
between amide N–H
groups and C=O
groups four residues
away.

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The α-keratins The α-keratins

association of
nonpolar side chains
between the two coils Major proteins of mammalian hair, nails, wool,
‘buries’ the
hydrophobic residues claws,horns, hooves, quills, beaks and outer
skin.

1° structure of helical rods consists of 7-residue

repeats: (a-b-c-d-e-f-g)n, where a and d are nonpolar

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The α-keratin: the structure of hair Collagen

- found in connective tissue (tendon,
cartilage)
- Collagen fibers form the matrix (or
cement) material in bones

- helical ("α-chain"), but not an α-helix

- left-handed, 3.3-residues per turn
- repeating tripeptide sequence
Gly-X-Pro (or Gly-X-HyPro)
- three helical strands wrap around each
other in a right hand sense to form a
'superhelix‘, with H bonds extending
between the chains

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Collagen molecules cluster to form fibrils Silk fibroin

• fibroin is a protein produced by spiders & insects
• spider dragline silk is both flexible and strong (tensile
strength of 200,000 psi
• structure is largely "β-sheet”

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Antiparallel β-sheets of silk fibroin Globular vs. Fibrous proteins

Globular Fibrous

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Tertiary Structure
 Three-dimensional arrangements of all the
atoms in the molecule.
 Polypeptide chain with its regions of secondary
structure, α-helix, and β-pleated sheet, further
folds on itself.
 Tertiary structure forms spontaneously and is
maintained as a result of interactions among
the R-groups of the amino acids.
 Globular protein like myoglobin has tertiary
structure
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R-group Interactions in Protein
Hydrophobic Interaction
Hydrogen bonding = formed between the H of
one molecule and O or N of the other molecule
Disulfide bond = sometimes formed between
two cysteine residues; plays a special role in
the structure of many proteins particularly those
that function extracellularly such as insulin and
immunoglobulins
 Ionic Interactions = occur between oppositely
charged groups; significant in determining the
shape and function of biomolecules.
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Protein Folding Patterns (folds, motifs) Myoglobin Structure

Without the oxygen (deoxymyoglobin), the remaining

coordination site on the other side of the iron is occupied
Polypeptide chain is often locally folded into one or by a water molecule. In the presence of oxygen
another of the kinds of secondary structure. (oxymyoglobin), this site is occupied by oxygen.

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Quaternary Structure Nomenclature for Quaternary Structure

• Quaternary structure is the result of noncovalent

interactions between two or more protein chains.
• In some cases the quaternary structure involves
binding to a nonprotein group called a prosthetic
group.
• Hemoglobin has four protein chains and the heme
prosthetic group.
• The chains in a quaternary structure may be identical
or different.
dimer = 2 polypeptide chains
trimer = 3 polypeptide chains
tetramer = 4 polypeptide chains

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Nomenclature for Quaternary Structure Themes in Protein Function

• structure dictates function

– primary structure → tertiary structure → protein
function

• proteins are often flexible & dynamic

– proteins have a unique shape & architecture, but many
(aside from certain fibrous/structural proteins) are not
rigid & immobile

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Themes in Protein Function
protein activity requires interactions with other
molecules
- a typical description is that of a protein-ligand
interaction
- a 'ligand' can range from being a small molecule (such
as an organic substrate for an enzyme) or a large one
(such as another protein, or DNA, etc.)
- the binding site is the region on a protein that a ligand
binds to (a protein may have more than one such site
and bind different ligands) (the binding site for an
enzyme is called the active site or catalytic site)
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Themes in Protein Function
• binding of a ligand to a protein often results in a
conformational change in one or both of the
interacting molecules
– induced fit is when a ligand binds to a protein, which
responds by changing its conformation to enhance
favorable binding interactions with the ligand
– for multisubunit proteins, a change in conformation in
one subunit can be 'felt' by the other subunits and
result in their conformational changes
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Oxygen-binding Proteins Structures of Myoglobin & Hemoglobin

•hemoglobin and myoglobin are heme-containing
oxygen transport and storage proteins (not enzymes)
– Hemoglobin (Hb) in blood
– Myoglobin (Mb) in muscle Each of the four chains
in in hemoglobin has a
• hemoglobin and myoglobin have different oxygen folded structure similar to
binding curves that of myoglobin and
each carries a heme.
• Mb is monomeric
– 153 aa, 16.7 kDa
• Hb is tetrameric (α2 β2) 64.5 kDa
– 2 α-subunits of 141 residues
– 2 β-subunits of 146 residues

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Heme: an oxygen-binding cofactor Heme-iron-protein interaction

Fe2+ is octahedrally coordinated. Four binding groups

from the porphyrin ring are attached to it. The remaining
- flat porphyrin ring system coordination sites lie along an axis perpendicular to the
- Fe2+ has six bonding sites ring. One coordination of these sites is occupied by the
nitrogen of His93 (proximal histidine).

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O2 versus CO binding to heme Hemoglobin (Hb)

• Hb is a tetrameric heme protein

• the basic physiological function of Hb is to bind oxygen

in lungs and release it in capillaries

• when a first oxygen binds to Fe in heme of Hb, the

heme Fe is drawn into the plane of the porphyrin ring
– Hb without a bound O2 molecule is called
deoxyhemoglobin

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Hemoglobin (Hb)
• this initiates a series of conformational changes that
are transmitted to adjacent subunits
• adjacent subunits' affinity for oxygen increases
• this is called positive cooperativity
• the Hb tetramer exists in one of two conformational
states:
– T state ('tense'; stabilized by more ion pairs) has low
O2 affinity
– R state ('relaxed') has high O2 affinity
– binding O2 changes the protein from T→ R
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Oxygen binding of hemoglobin
• Oxygen binds to heme in hemoglobin
cooperatively: as one O2 is bound, it becomes
easier for the next to bind.
• The first oxygen causes 2,3-bisphospho-glycerate
(BPG) to leave deoxyhemoglobin. This causes
shape changes which favor more reaction with
oxygen.
• H+ produced by metabolizing cells (low pH) favors
oxygen release from Hb and higher pH in the lungs
favors binding of oxygen to Hb.
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pH/[CO2] variation & O2-binding to Hb Oxygen Transport: Mother → Fetus

• Hb can bind H+ and CO2 and • Fetus receives its oxygen from its mother by simple
serves to carry them from the
diffusion across the placenta.
tissues where they are
generated to the lungs & • Fetus has a unique type of hemoglobin called fetal
kidneys where they are excreted hemoglobin.
• at low pH (high [H+]) and
• This Hb has greater affinity for oxygen than does the
high [CO2] Hb has reduced
O2 affinity mother’s Hb because it does not bind BPG as well as
• O2 & H+ have inverse binding adult Hb.
affinities to Hb • Biosynthesis of fetal Hb stops shortly after birth when
at [O2] - releases H+ from Hb the genes coding fetal Hb are switched off and the
(as in lung)
at [O2] - H+ binds Hb, O2
genes coding for adult Hb are switched on.
released (as in peripheral tissue)

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Proteins in Muscle Protein Denaturation

 Major proteins in muscle: actin & myosin
 Actin exists as long, helical polymer of a globular Denaturing destroys the physiological
protein monomer (G-actin monomer). function of the protein (remove the 2o-4o
 Myosin is composed of six polypeptide chains. interactions).
Heat (coagulation)
Change in pH leads to coagulation or
unfolding to to charge repulsion
Heavy metals
Detergents
Organic solvents

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Protein Digestion and Diet Meat Protein Categories

Digestion:
Stomach: pepsin  Myofibrillar proteins (contractile)
S. I.: trypsin, chymotrypsin, elastase, etc.
Essential Amino Acids:
Cannot be synthesized by humans  Stromal proteins (connective)
Include: Ile, Leu, Lys, Met, Phe, Thr, Try, Val
Complete protein from animals provides esential AA
in proper proportions.  Sarcoplasmic proteins (water soluble,
Incomplete protein from vegetable sources must be intracellular fluid)
balanced. E. g. beans (lys + trp) and corn (met)

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Categories
Myofibrillar proteins

 ~ 55% of total muscle protein

 critical to processing properties

 critical to processing properties

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Components of myofibrillar Myosin

proteins
Myosin is generally considered the
myosin ~55%
singly most important because:
actin
troponin 40 - > Long filamentous molecule
tropomyosin 45% (similar to a 1 inch garden hose
that is 8 feet long)
desmin, synemin,  > Amino acid composition gives
actinin, nebulin and 1- highly-charged, polar molecule
numerous structural 5% > Present in large quantity in lean
proteins muscle

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Other proteins are also important
> Many are charged, polar molecules
> Structural proteins can have a
large influence on “release” of
myosin/actin and “opening”
protein structure to water.
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Stromal proteins
 ~ 10-15% of total muscle protein
 Primarily collagen
most abundant protein in animal body (20-
25% of total protein) – skin, sinews, tendons, etc.
 Designed to transmit force and hold
things together, therefore, these
proteins are generally tough and
inert
 Increased crosslinking as animal age
increases toughness and major cause
for sausage and ground beef industries
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Stromal proteins Stromal proteins

 Not very valuable in processed meats --
Collagen
- has little binding ability

Will shrink when heated to 140oF+ (with

moisture) and convert to gelatin at 160oF
- 180oF
- but - if heated when dry --- collagen
becomes very hard and impermeable ---
important to handling of collagen and/or
natural casings

- collagen is highly resistant to enzymes

so enzyme tenderizers are generally
ineffective

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Stromal proteins
 Collagen is used to make gelatin,
contact lenses, and sausage
casings
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Stromal proteins
 Unique protein with
~ 33% glycine
~ 10% hydroxyproline
therefore very nonpolar and noncharged
molecule - isoelectric point is pH 7.2
 By far the only protein to contain
large amount of hydroxyproline
hydroxyproline measurement is the
most common method used to
determine collagen content in meat
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Sarcoplasmic proteins Importance of Sarcoplasmic proteins

1. Enzyme activity
 ~ 30% of total muscle protein (~20% –calpain - tenderization
of binding ability) –postmortem glycolysis
–potential flavor contributions from
 Isoelecteric points generally between protein hydrolysis  hydrolyzed
pH 6-7. proteins

 Most are relatively low molecular

weight (small) proteins 2. Color
–myoglobin
–responsible for all meat color variations
so a good understanding is critical in
meat processing

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