PROTEIN STRUCTURE AND

FUNCTION

BY
MR. JOHN OMARA
0772854603
[email protected]
 Chapter 1

Amino acids, peptides, and

proteins
 Properties of Amino Acids
• capacity to polymerize
• novel acid-base properties
• varied structure and chemical
functionality
• Chirality
• Vary in size, shape, charge, hydrogen
bonding capacity, chemical reactivity
 Basic Amino Acid Structure
carboxyl group

-carbon is
chiral (except for amino group
glycine)
 at pH 7.0
uncharged amino
acids are -carbon
zwitterions side chain

 amino acids have

a tetrahedral
structure
Amino Acid Enantiomers

•Steroisomers / enantiomers
•Biological system only synthesize and
use L-amino-acids (22 proteinogenic
amino acids)
 Amino Acid Classification
• Aliphatic
• Aromatic
Hydrophobic
• Sulfur containing
• Selenium containing
• Polar/uncharged Hydrophillic
• basic/acidic
 Aliphatic (alkane) Amino Acids
•Proline (pro, P)- cyclic “α-imino acid”
Hydrophobicity

•Glycine(gly, G)-only non-chiral amino acid, not hydrophobic

•Alanine (ala, A) – R-group = methyl-group

•Valine (Val, V) –Think V!

•Leucine (Leu, L) –

•Isoleucine (Ile, I) -2 chiral carbons

 Aromatic Amino Acids
• All very hydrophobic
• All contain aromatic group
• Absorb UV at 280 nm
• Phenylalanine (Phe, F)
• Tyrosine (Tyr, Y) – -OH ionizable (pKa = 10.5), H-Bonding,
least soluble amino acid (0.453g/L at 250C)
• Tryptophan (Trp, W) – bicyclic indole ring, H-Bonding
 Sulfur Containing Amino
Acids
• Methionine (Met, M) – “start” amino
acid, very hydrophobic

• Cysteine (Cys, C) – sulfur in form of

sulfhydroyl, important in disulfide
linkages, weak acid, can form
hydrogen bonds.
 Selenium containing
• Selenocystein (Sec, U)
• Recognised as the 21st amino acid
• Has both a lower pKa and a higher
reduction potential than cysteine
• No free pool of selenocysteine exists
in the cell
 Acidic Amino Acids
• Contain carboxyl groups (weaker acids than a-carboxyl-
group)
• Negatively charged at physiological pH, present as conjugate
bases (therefore –ate not –ic acids)
• Carboxyl groups function as nucleophiles in some enzymatic
reactions
• Important in metal binding in metalloproteins
• Aspartate (Asp, D) Glutamate (Glu, E)
 Basic Amino Acids
• Hydrophillic nitrogenous bases
• Positively charged at physiological pH
• Histidine (His, H) – imidazole ring protonated/ionized, only
amino acid that functions as buffer in physiological range,
accepts and donate protons in enzymatic reactions.
• Lysine (Lys, K)- diamino acid, protonated at pH 7.0
• Pyrrolysine (Pyl, O) 22nd amino acid, found in methanogenic
archaea and one known bacterium
• Arginine (Arg, R) - guianidinium ion always protonated, most
basic amino acid
Polar Uncharged Amino Acids
• Polar side groups, hydrophillic in nature, can form hydrogen
bonds

• Hydroxyls of Ser and Thr weakly ionizable

• Serine (Ser, S) – looks like Ala w/ -OH

• Threonine (Thr, T) – 2 chiral carbons

• Asparagine (Asn, N) – amide of aspartic acid

• Glutamine (Gln, Q) – amide of glutamic acid

 Uncommon amino acids
Amino acid Protein
Hydroxyproline & hydroxylysine Collagen and gelatin
Thyroxine & 3,3,5-triiodothyronine Thyroglobulin
Methylhistidine, e-N-methyllysine and e- Certain muscle protein
N,N,N-trimethyllysine

γ-Carboxyglutamate Prothrombin, Ca2+ binding proteins

Pyroglutamic acid Bacteriorhodopsin
Phosphorilated proteins Proteins involved in cell growth and
regulation

Amino adipic acid Corn proteins

N-methylarginine and N-acetyllysine Histones
6-N-Methyllysin Myosin
 Amino acids not found in proteins
Amino acid Function

γ-aminobutyric acid (GABA) Neurotransmitter

Histamin and serotonin Neurotransmitters & regulators

β-alanine Component of carnosine, anserine &

pantothenic acid

Penicillamine Constituent of penicillin antibiotic

Ornithine, betamine, homocystein Metabolic intermediates

&homoserine

Citruline Precursor of arginine

 Reactions of amino acids
• α- amino & α-carboxyl gps exhibit similar reactivity
• Different reactivities and biological functions of amino acids are
due to various functional groups in side chains
• React with ammonia and primary amines to form unsubstituted
and substituted amines respectively
• Esterification in presence of alcohol and strong acid
• Polymerization
• Reaction with acid chlorides
• Free amino gps react with aldehydes forming schiff bases
• Ninhydrin reaction forming purple color, proline & hydroxyproline
give bright yellow products. Used on fingerprinting
• Cystein residues form disulfide bonds, also rxt with N-
ethylmaleimide, iodoacetic acid to yield S-carboxymethyl cystein
Spectroscopic properties of aas

• No amino acid absorbs light in visible range

• All absorb in infrared range

• Aromatic aa (Phe, Tyr &Trp) absorb in uv range above

250nm

• Aromatic aa exhibit fluorescence and phosophoresence;

useful in study of protein structure and dynamics

• NMR used in chemical characterization of amino acids

Essential/Non-Essential Amino Acids

• Essential –histidine, isoleucine,

leucine, lysine, methionine,
phenylalanine, threonine, tryptophan,
valine
• Non-essential – alanine, arginine*,
aspartate, asparagine, cysteine*,
glutamate, glutamine, glycine*,
proline*, serine, tyrosine*
 Protein Nomenclature

Constitute > 50% of dry weight of cells

• Peptides 2 – 50 amino acids
• Proteins >50 amino acids
• Amino acid with free -amino group is the
amino-terminal or N-terminal residue
• Amino acid with free -carboxyl group is the
carboxyl-terminal or C-terminal residue
• Three letter code – Met-Gly-Glu-Thr-Arg-His
• Single letter code – M-G-E-T-R-H
Peptide Bond Formation
Stability and Formation of the Peptide Bond

• Hydrolysis of peptide bond favored energetically,

but uncatalyzed reaction very slow.
• Strong mineral acid, such as 6 M HCl, good
catalyst for hydrolysis
• Amino acids must be "activated" by ATP-driven
reaction to be incorporated into proteins
 Enzymatic and Chemical
Cleavage of Peptide Linkage
 Chapter 2

Protein 3-Dimensional Structure and

Function
 Terminology
• Conformation – spatial arrangement
of atoms in a protein
• Native conformation – conformation
of functional protein
 Protein Classification
• Simple – composed only of amino acid residues e.g ribonuclease, actin
• Conjugated – contain prosthetic groups
Glycoproteins: contain carbohydrates, extracellular proteins e.g
frbronectin, proteoglycans, IgG, membrane proteins
Lipoproteins: contain lipid, eg HDL, LDL. Low levels of LDL used as clinical
index for susceptibility to vascular disease
Nucleoproteins: contain nucleic acid, storage &transfer of genetic
information, e.g ribosomes, HIV-1, Adenovirus
Phosphoproteins: contain phosphate gps, e.g casein, glycogen phosphorylase
a
Metalloproteins: either storage eg ferritin (Fe), or enzymes eg alcohol
dehydrogenase (Zn), cytochrome oxidase (Cu, Fe), nitrogenase (Mo, Fe),
pyruvate carboxylase (Mn)
 Protein Classification
• One polypeptide chain - monomeric protein
• More than one - multimeric protein
• Homomultimer - one kind of chain
• Heteromultimer - two or more different
chains

(e.g. Hemoglobin is a heterotetramer. It has

two alpha chains and two beta chains.)
 Protein Classification
Fibrous –
1) polypeptides arranged in long strands or
sheets
2) water insoluble (lots of hydrophobic AA’s)
3) strong but flexible
4) Structural (keratin, collagen [1/3 total protein
in verterbrate animal])
Globular –
1) polypeptide chains folded into spherical or
globular form
2) water soluble
3) contain several types of secondary structure
4) diverse functions (enzymes, regulatory
proteins)
catalase keratin

Collagen (Gly-X-
Pro repeat units)
 Protein Function
• Catalysis – ribonuclease, trypsin, catalase
• Structural – keratin, collagen, elastin, fibroin, proteoglycans
• Transport – hemoglobin, serum albumin, Na+/K+ ATPases
• Toxins – rattle snake venom, ricin
• Contractile & motile function – actin, myosin, tubulin, dynein,
kinesin
• Regulatory – insulin, lac repressor
• Scaffold – Grb2, crk, shc, stat, IRS-1
• Storage Proteins – casein, zein, ovalbumin, ferritin, phaseolin
• Defensive & exploitative proteins – Immunoglobulins, thrombin,
fibrinogen, antifireze proteins, venoms, ricin
• Exotic proteins: monellin, resilin, glue proteins
4 Levels of Protein Structure
 Non-covalent forces
important in determining
protein structure

• van der Waals: 0.4 - 4 kJ/mol

• hydrogen bonds: 12-30 kJ/mol
• ionic bonds: 20 kJ/mol
• hydrophobic interactions: <40 kJ/mol
 1o Structure Determines 2o, 3o, 4o
Structure
• Sickle Cell Anemia – single amino
acid change in hemoglobin related
to disease
• Osteoarthritis – single amino acid
change in collagen protein causes
joint damage
 Protein denaturation
• Denaturation – disruption of native
conformation
• Heat commonly used to denature
proteins
• Tm = temperature where 50%
folded/50% unfolded. Tm
• Typical Tm = 40-60oC
• Tm for thermophiles >100oC
(Taq DNA polymerase)
• Chemical denaturants Chaotrophic
agents = Urea, KCN detergents = SDS
 Disulfides Bonds
• Stabilize native structure
• Formed after native conformation achieved
• Abundant in secreted proteins but not in intracellular
proteins
• Protein disulfide isomerase catalyzes reduction of
incorrect disulfide linkages
 Chapter 3

Protein purification and Analysis

 Why purify proteins?
• Pure proteins are required to study
enzyme function
• Pure proteins are required for structural
analysis (x-ray crystallography, NMR
spectroscopy)
• Pure proteins are required to obtain amino
acid sequence
Steps in protein purification
• Develop assay
• Choose source of protein
• Prepare tissue extract
– cell disruption
– subcellular fractionation
• Protein fractionation (several steps)
• Determination of purity
 Differential Centrifugation
transfer transfer transfer
supernatant supernatant supernatant

1000 g 10,000 g 100,000 g

Pellet Pellet Pellet Super.

tissue
unbroken cells mitochondria microsomal Cytosol,
homogenate
nuclei Fraction Soluble
chloroplast (ER, golgi, enzymes
lysosomes,
 Protein precipitation
• Protein solubility depends on pH and
ionic strength
• Least soluble at pI
• Ionic strength influence solubility of
globular proteins
• Salts like ammonium sulphate used
Chromatography
Gel Permeation Chromatography
 Gel Permeation
Chromatography
Ion-exchange Chromatography
 Ion-exchange
Chromatography
+++ +++
- - - low salt buffer high salt buffer
- - - - - -
+ + +++ ++ + -+
Cl
++++ + ++ + +-
-+ -
+ + - + ++ Cl+- + +- + Cl-+
+ +-
+ ++ +++ + + ++ - -
+ ++ + Cl
+ + +
+ +Cl+
+ ++ + - - - ++ +
+
Cl+- + +
+
+ + +
+ + + + + +
+++ + ++ +++ + ++ +++ + ++
+ + + + + +
+ +++ + + +++ + + +++ +
+ ++ + + ++ + + ++ +

+++ +++ - - - - - -
+++ - - -
Affinity Chromatography
Affinity Chromatography
Add excess ligand
SDS poly acrylamide electrophoresis (PAGE)
SDS = H3C-(CH2)10-CH2-OSO3-
SDS – denatures protein
coats w/
- negative charge
--- --
- -- - -- --
--

Used to determine protein MW

And purity of protein prep
 Isoelectric Focusing
pH 9
- -
Decreasing pH

Decreasing pH

pH 3
+ +
 2-D Electrophoresis
Decreasing pH
large
Decreasing MW

Decreasing MW
SDS-PAGE

small
+
Decreasing pH
 Protein sequencing
• Protein chains are separated and purified
• Disulfide bonds cleaved
• Amino acid composition determined
• N-terminal and C-terminal residues identified
• Polypeptide chain enzymatically cleaved to
generate overlaping fragments
• Fragments purified and sequenced (Edman
degradation, Tandem mass spectrometry)
• Overlall aa sequence reconstructed
• Positions of S-S bonds located
 Peptide mapping exercise
Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-As

Trypsin Chymotrysin
Met-Ala-Arg Met-Ala-Arg- Gly-Glu-Tyr
Phe-Ala-Glu-Gln-Asp Met-Cys-Lys –Phe
Gly-Glu-Tyr-Met-Cys- Ala-Glu-Gln-Asp
Lys CNBr
Met
Ala-Arg-Gly-Glu-Tyr-Met
Cys-Lys-Phe-Ala-Glu-Gln-Asp
 Sequence databases
• Protein sequences can also be obtained from DNA
sequences using genetic code
• Atlas of protein sequence and structure
• Protein Identification Resource (PIR), Genetic
Sequence Data Bank (GenBank), Europeam
Molecular Biology Laboratory Data Library (EMBL)
 Importance of Protein
Sequencing
• Explanation of molecular pathology of diseases eg.
Sickle cell anaemia
• Understanding molecular basis of biological
activity
• Determining evolutionary history
 References
• Biochemistry, by L Stryer
• Basic Neurochemistry: Molecular,
Cellular and Medical Aspects. 6th
edition. By Siegel, et. al., editors.
• Principles of Biochemistry, by Lehninger
et. al.
• Clinical chemistry: Theory, analysis and
correlation, by Kablar and Pesce