Structure & Function

DNA mRNA Protein

* Reminder: quiz next week

 More than 300 amino acids have been described in
nature.

 20 are commonly found as constituents of

mammalian protein.
 the only amino acids coded for by DNA.
 Extend the biologic diversity of proteins.
 Altering the solubility, stability, and interaction with
other proteins.
 proline and lysine : 4-hydroxyproline and 5-
hydroxylysine
 The conversion of glutamate to γ-carboxyglutamate
 The methylation, formylation, acetylation,
prenylation, and phosphorylation of certain
aminoacyl residues.
 Consist of:
 Carboxyl group (organic acid).
 A primary amino group (organic base).
 Side chain (R-group) bonded to the carbon center (α-
carbon).

 Also called α-amino acids (fx gp around α-carbon).

Structural features of amino acids (shown
in their fully protonated form).
 α-carbon is attached to 4 different chemical groups:
 Asymmetric center
 A chiral carbon (optically active carbon atom-rotate the angle of
plane-polarized light).

 Glycine
 Exception
 Has 2 hydrogen substituents (R=H)
 Optically inactive
 Exist in 2 forms:
 Dextrorotatory & levorotatory.
 D and L : mirror image of each other.
 Stereoisomers, optical isomers or enantiomers.
 Drawing to show absolute
configuration:
 Carboxyl group: drawn up &
behind the page
 R group: down, behind

 Therefore:
 D-isomer: aa gp in front, to
the right
 L-isomer: front, left
 D & L isomers

 Both exist in nature.

 L isomers
 Used as building blocks for all proteins .
 Microorganisms
 peptides that contain both D- and L-α-amino acids.
 therapeutic value – eg: antibiotics bacitracin and
gramicidin A, antitumor agent bleomycin.
 Some are toxic. The cyanobacterial peptides microcystin
and nodularin are lethal in large doses, while small
quantities promote the formation of hepatic tumors.
 D & L isomers

 D-amino acids in human:

 Brain
▪ neurotransmitter
 Human teeth, lenses, erythrocytes, some tumors.
All 20 -amino acids in pure form
 White, crystalline, high-melting solid.
 Soluble in water, insoluble in organic solvents (acetone,
ether, chloroform).
 Aq solutions: conduct electric current
Physiologic pH

 carboxyl group dissociate:

 Negatively charged
carboxylate ion (_COO-).
 Donate H+.

 Amino group (-NH2)

 Basic
 Able to accept proton:
protonated amino group
(_NH3+).

 R side chains
 May have acidic/basic fx
groups
 Donate/accept electron
 Dipolar ionic species: +ve & -ve charge
 Zwitterions
 Assuming R has no charge
▪ Net charge of zwitterion = 0
 Amino acids in aqueous solution

 contain weakly acidic α-carboxyl groups

 weakly basic α-amino groups.
 Can act as buffers.
 Amino acids are weak acids.
 At least 2 titratable protons
 α-carboxyl (-COOH)
 α-amino (-NH3+)
 2 pKa’s
 Some have 3rd titratable proton
 R group
 3rd pKa
 The protonic equilibrium of ionizable -COOH
and -NH3+ :
 Amino acids may have positive, negative, or zero
net charge.

 Structure of amino acid change depends on pH.

 Isoelectric pH (pHI)
 pH where molecular structure has no net charge
 Properties of amino acids in proteins and peptides
determined by
 R group
 charges of the titratable group.
 Both affect protein structure.
 Important to know which groups on peptides and
proteins will be protonated at a certain pH.
Fully protonated structure of
alanine?
 Which protons come off when?
 pKa table for amino acids
 α-COOH (pKa = 2.3) comes off first (has lower pKa)
 α-NH3 + (pKa = 9.9)
 At different pH’s, amino acids can have
different charges
 Very important for protein structure
 2 midpoints (pKa’s) – one for each proton α-COOH
and α-NH3+
 Start with all protonated.
 Need one equivalent of base for each proton
 - At end all deprotonated
 Flat parts of curve are BUFFERING REGIONS
 Acts as buffer in TWO pH ranges.
 o +/- 1 pH unit from pKa
 To determine whether the proton is ON or OFF at a
certain pH:
 pH = pKa : equal amounts of protonated and deprotonated
species exist
 If pH is LESS than the pKa of a particular group
 That group will be predominantly protonated

 if pH is GREATER than the pKa of a particular

ionizable group
 that group will be predominantly deprotonated
 At pH 1.5: pH is less than the pKa of
both the α-COOH and the α-NH3+,
therefore, both protons are ON
pH is greater than the pKa of the α-COOH  H+
OFF
pH is less than the pKa of the α-NH3+  H+ ON
 Distinction (chemically/physically) of amino acids: R

 R grp varies in
 Size, polarity, charge, chemical reactivity.

 Classification: polarity & reactivity of R.

Side chain

 aliphatic/aromatic gp containing
C-H
 nonpolar.

 lipid-like property - hydrophobic

interaction.

 Does not
 gain/loose protons
 participate in hydrogen/ionic bonds.

 Little reactivity.
Location:

 Proteins in aqueous
solutions
 cluster in the interior of the
protein (hydrophobic effect).

 Hydrophobic environment
 outside surface of the protein.
Glycine

 R=H

 How to classify?

 Unreactive nature-similar
to nonpolar amino acids
Proline

 Differ from other amino acid.

 Proline’s side chain and α-amino N

form a five-membered ring structure.

 Has secondary amino group.

 Imino acid.
 Unique geometry contributes to the
formation of the fibrous structure of
collagen.
 Variety of functional gp,
with heteroatom (N, O, S)
 Got electron pairs available for
hydrogen bonding with
H2O/other molecules.

 Eg: cysteine

 Has -SH (thiol/sulfhydryl

group)

 -SH can react with –SH of

another cysteine
 Disulfide bond (oxidizing agent
present)
 Side chain (imidazole).
 pKa value 6.0
 relatively small shifts in pH will change its average charge.
 Below pH of 6, the imidazole ring is mostly protonated.
 2 major ionic forms: depend on in vivo conditions.
 pH 7.4: slight abundance of zwitterionic form.

pKr – pKa of the R gp

 Functional gp of side chain-acidic/basic.
 Aspartate, glutamate: the side chains are fully
ionized (physiologic pH)
 Proton donors
 Negatively charged carboxylate group (_COO-).
 The names emphasized on the acidic properties.
 Lysine
 Side-chain: amino group
 Arginine
 Guanidino group
 Lysine & arginine: +1 ionic state
Titration curve
 For gp III, 3 pKa values
 pK1: -carboxyl group
 pK2: -amino group
 pKR: ionizable group on the R side chain
 Carboxyl groups, can be converted to
 Esters, amide
 Linked to other amino acid-form amide/peptide bond
 Amino groups
 Amide
 Side chain hydroxyl groups
 React with acids-ester products
Glutamate
 Proteins sometimes contain amino acids that are
not in the set of 20.
 Derived by chemical reactions on standard amino
acids.
 Eg:
 Chemical modification of
▪ tyrosine to O-phosphotyrosine
▪ Serine to phosphoserine
▪ Regulate enzymes activities
 Method:
 Degrade the protein into its individual amino acids.
 Separate the amino acids.
 Identify and measure the amino acids

 Method to separate, identify & measure:

chromatography
Peptide Linkages

 Two amino acids can be linked

together by
 Amide/peptide bond
 Reaction:
 Condensation (loss of a H2O
molecule)
 Between carboxyl gp of one amino
acid with amino gp of other.
 Form a dipeptide
 2 to 10 acid amino residues,
prefix for numbers: tri-, tetra-,
penta-, hexa-, hepta-, octa-,
nona-, decapeptide).
 10-100: polypeptides
 >100 : proteins
 2 distinct end:
 Amino terminus (N-terminus).
 Carboxyl terminus (C-terminus).

 Numbering of peptides:
 N-terminus to C-terminus

 -amino & -carboxyl of each

amino acids (except for terminus)
not available for ionization.

 R side chains for each residue

remain unchanged
N-terminus C-terminus
 Important role in protein folding
1 2 3
 Short/long polypeptide.

 Glutathione
 Tripeptide: glutamic acid, cystine, glycine
 Regulate oxidation-reduction reactions
 Destroy free radicals.

 Insulin
 51 amino acids.
 hormone
 Catalyst.
 Facilitate biochemical reactions.
 Eg:
 Amylase: digestion of CHO in the diet.
 DNA polymerase: DNA replication
 Provide mechanical support to
cells/organism.

 Proteins of intracellular cytoskeleton

 Assemble actin filaments, microtubule,
intermediate filaments.
 Defense purposes.
 Antibodies, proteins that selectively bind and
neutralize foreign substances.
 Smaller biomolecules need carrier to be transported
throughout an organism
 O2
 Cholesterol
 Fatty acids

 Protein-carrier: through blood stream, across

membrane

 Hb, lipoprotein, specific membrane protein

 Regulate cellular & physiological activities.

 Hormones.

 G protein: transmit hormonal signals inside

the cells.

 DNA-binding protein.
 Components of the contractile system of
skeletal muscle
 Actin & myosin

 Movement of sperm & protozoa by flagella &

cilia
 Depends on protein dynein.
 Calculated by summing the molecular mass
of the amino acid residues.

 Estimation
No of aa x average molecular weight (110) = daltons (Da)

1 daltons = 1 atomic mass unit

Eg: protein with 250 aa: molecular mass of 27 500 Da/27.5 kDa
 Monomeric
 A single polypeptide chain
 Eg: cytochrome c

 Oligomeric
 2 or more polypeptide chain.
 Held by noncovalent interaction

 Subunit/multisubunit
 Each peptide is referred as subunit.
 May be identical/different
 Eg: Hb-tetrameric
▪ 4 subunit: 2 α-type chain, 2 -type chain
 Protein Composition & Behaviour

 Simple proteins
 Only acid amino residues, no other biomolecules.
 Eg: trypsin, chymotrypsin

 Conjugated proteins
 Contain other chemical groups: small organic molecule,
metal atom
 Additional chemical group: prosthetic group
 Eg: Hb-each subunits have a heme prosthetic group
containing iron.
 Eg: alcohol dehydrogenase: 4 subunit, each associated
with zinc atom.
Protein Composition & Behaviour

 Nature of R determine the physical properties of

protein.

 Neutral pH
 +ve charge N terminus, -ve C terminus neutralized each
other.

 Important when protein is characterized by

electrophoresis or solubility
 Purify, measuring the molecular size of proteins.
 Protein Composition & Behaviour

Categorized base on solubility:

 Globular proteins
 Water soluble
 Relatively high content of residues with polar and charged R groups.
 Important in transport, immune protection, catalysis etc.
 Dissolved in biological fluid: blood, cytoplasm
 Form ordered, but dynamic & flexible conformation.

 Fibrous protein
 Water insoluble.
 Structural proteins: collagen, keratin
 Residues with nonpolar R groups.
 Form ordered and rigid conformation.
 Amino acids are joined together by peptide
bond.

 4 organizational level of proteins:

 Primary
 Secondary
 Tertiary
 quartenary
 The sequence of amino
acids in a protein.

 Amino acids are joined

covalently by peptide bond
 Linkage between α-carboxyl
group of one amino acid and
the α-amino group of other.
 Not easily broken by
conditions that denature
protein.
 Free amino end (N-
Same figure
terminal) of the
peptide chain is written
to the left.

 Free carboxyl end (C-

terminal) to the right.
Noncovalent interactions
 important forces that stabilize protein three-
dimensional structure
 Hydrogen bonding between
 atoms of acid amino residues
 Atoms of acid amino residues with H2O.

 Ionic bonds
 Van der Waals forces
 Regular rearrangements of amino acids located
near each other in the linear sequence.

 Secondary structure:
 α-helix
 -sheet
 -bends

 Nonrepetitive secondary structure.

 Supersecondary secondary structure.

 Folding of domains (the basic units of
structure and function)
 to the final arrangements of domains in
polypeptide.

 Hydrophobic side chains: interior.

 Hydrophilic group: surface of the molecule.

 Two or more protein subunits(structurally
identical/totally unrelated)
 arranged & held together
 noncovalent interactions
▪ hydrogen bonds, ionic bonds, hydrophobic interactions).

 May work indepedently or cooperatively.

 Eg: hemoglobin