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amino asid,peptides and proteins
1. CHAPTER 3
Amino Acids, Peptides,
Proteins
• Structure and naming of amino acids
• Structure and properties of peptides
• Ionization behavior of amino acids and peptides
• Purification and assay methods
• Peptide sequencing and chemical synthesis
• Protein sequence analysis
2. Proteins: Main Agents of
Biological Function
• Catalysis:
–enolase (in the glycolytic pathway)
–DNA polymerase (in DNA replication)
• Transport:
–hemoglobin (transports O2 in the blood)
–lactose permease (transports lactose across the cell membrane)
• Structure:
–collagen (connective tissue)
–keratin (hair, nails, feathers, horns)
• Motion:
–myosin (muscle tissue)
–actin (muscle tissue, cell motility)
3. Amino Acids: Building Blocks of
Protein
• Proteins are heteropolymers of α-amino acids
• Amino acids have properties that are well
suited to carry out a variety of biological
functions:
– Capacity to polymerize
– Useful acid-base properties
– Varied physical properties
– Varied chemical functionality
4. Amino Acids: Atom Naming
• Organic nomenclature: start from one end
• Biochemical designation: start from
α-carbon and go down the R-group
5. Most α-Amino Acids are Chiral
• The α-carbon has always
four substituents and is
tetrahedral
• All (except proline) have an
acidic carboxyl group, a
basic amino group, and an
alpha hydrogen connected
to the α-carbon
• Each amino acid has an
unique fourth substituent R
• In glycine, the fourth
substituent is also hydrogen
6. Amino Acids: Classification
Common amino acids can be placed in five
basic groups depending on their R substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)
13. Not incorporated by ribosomes
Arise by post-translational modifications of
proteins
Reversible modifications, esp.
phosphorylation is important in regulation
and signaling
Uncommon Amino
Acids in Proteins
15. Ionization
At acidic pH, the carboxyl
group is protonated
and the amino acid is
in the cationic form
At neutral pH, the
carboxyl group is
deprotonated but the
amino group is
protonated. The net
charge is zero; such
ions are called
Zwitterions
At alkaline pH, the amino
group is neutral –NH2
and the amino acid is
in the anionic form.
16. Substituent effects on pKa Values
α-carboxy group is much more acidic than in carboxylic acids
α-amino group is slightly less basic than in amines
17. Amino Acids Can
Act as Buffers
Amino acids with
uncharged side-chains,
such as glycine, have two
pKa values:
The pKa of the α-carboxyl
group is 2.34
The pKa of the α-amino
group is 9.6
It can act as a buffer in
two pH regimes.
18. Amino Acids Carry a Net Charge
of Zero at a Specific pH
•Zwitterions predominate at pH values between the pKa values
of amino and carboxyl group
•For amino acid without ionizable side chains, the Isoelectric
Point (equivalence point, pI) is
• At this point, the net charge is zero
– AA is least soluble in water
– AA does not migrate in electric field
2
21 pKpK
pI
+
=
19. Ionizable Side Chains Can Show
Up in Titration Curves
• Ionizable side chains
can be also titrated
• Titration curves are
now more complex
• pKa values are
discernable if two pKa
values are more than
two pH units apart
Why is the side-chain
pK so much higher?
20. How to Calculate the pI When the
Side-chain is Ionizable?
• Identify species that carries
a net zero charge
• Identify pKa value that
defines the acid strength of
this zwitterion: (pK2)
• Identify pKa value that
defines the base strength of
this zwitterion: (pKR)
• Take the average of these
two pKa values
21. Peptides and Peptide bonds
Peptide bond in
a di-peptide
“Peptides” are
small
condensation
products of
amino acids
They are “small”
compared to
proteins (di, tri,
tetra… oligo-)
22. Peptide Ends are Not the Same
Numbering starts from the amino terminus
AA1 AA2 AA3 AA4 AA5
23. The Three Letter Code
• Naming starts from
the N-terminus
• Sequence is written
as:
Ala-Glu-Gly-Lys
• Sometimes the one-
letter code is used:
AEGK
24. Peptides: A Variety of Functions
• Hormones and pheromones:
– insulin (think sugar)
– oxytocin (think childbirth)
– sex-peptide (think fruit fly mating)
• Neuropeptides
– substance P (pain mediator)
• Antibiotics:
– polymyxin B (for Gram - bacteria)
– bacitracin (for Gram + bacteria)
• Protection, e.g. toxins
– amanitin (mushrooms)
– conotoxin (cone snails)
– chlorotoxin (scorpions)
25. Proteins are:
• Cofactor is a general term for functional non-amino acid component
– Metal ions or organic molecules
• Coenzyme is used to designate an organic cofactors
– NAD+
in lactate dehydrogenase
• Prosthetic groups are covalently attached cofactors
– Heme in myoglobin
• Polypeptides (covalently linked α-amino acids) + possibly –
• cofactors,
• coenzymes,
• prosthetic groups,
• other modifications
28. Peptides and Proteins-
Burning Questions
Sequence and composition?
Three-dimensional structure?
Folding Mechanism?
Biochemical role?
Functional regulation?
Molecular interactions with small and macro-molecules?
Structural and sequence relatives?
Cellular and sub-cellular localization?
Physical and chemical properties?
29. Purification – Fractionation of
Protein Mixtures
• Separation relies on differences in physico-
chemical properties
– Solubility – Selective Precipitation (Centrifugation)
– Thermal stability --
– Charge --Electrophoresis, Isoelectric Focusing, IEC
– Size – Dialysis, Sedimentation (Centrifugation), GFC
– Affinity for a ligand – “Pull down” assays (Centrifugation),
AC
– Hydrophobicity (HIC)
• Chromatography is commonly used for
preparative separation
34. Electrophoresis for Protein
Analysis
Separation in
analytical scale is
commonly done by
electrophoresis
– Electric field pulls
proteins according to
their charge
– Gel matrix hinders
mobility of proteins
according to their
size and shape
35. SDS PAGE: Molecular Weight
• SDS – sodium dodecyl
sulfate – a detergent
• SDS micelles binds to,
and unfold all the
proteins
– SDS gives all proteins an
uniformly negative
charge
– The native shape of
proteins does not matter
– Rate of movement will
only depend on size:
small proteins will move
faster
-
37. Spectroscopic Detection of Aromatic
Amino Acids
• The aromatic amino acids
absorb light in the UV
region
• Proteins typically have
UV absorbance maxima
around 275-280 nm
• Tryptophan and tyrosine
are the strongest
chromophores
• Concentration can be
determined by UV-visible
spectrophotometry using
Beers law: A = ε·c·l
38. Chapter 3: Summary
In this chapter, we learned about:
• The many biological functions of peptides and
proteins
• The structures and names of amino acids found in
proteins
• The ionization properties of amino acids and
peptides
• The methods for separation and analysis of
proteins
FIGURE 3-16 Column chromatography. The standard elements of a chromatographic column include a solid, porous material (matrix) supported inside a column, generally made of plastic or glass. A solution, the mobile phase, flows through the matrix, the stationary phase. The solution that passes out of the column at the bottom (the effluent) is constantly replaced by solution supplied from a reservoir at the top. The protein solution to be separated is layered on top of the column and allowed to percolate into the solid matrix. Additional solution is added on top. The protein solution forms a band within the mobile phase that is initially the depth of the protein solution applied to the column. As proteins migrate through the column, they are retarded to different degrees by their different interactions with the matrix material. The overall protein band thus widens as it moves through the column. Individual types of proteins (such as A, B, and C, shown in blue, red, and green) gradually separate from each other, forming bands within the broader protein band. Separation improves (i.e., resolution increases) as the length of the column increases. However, each individual protein band also broadens with time due to diffusional spreading, a process that decreases resolution. In this example, protein A is well separated from B and C, but diffusional spreading prevents complete separation of B and C under these conditions.
In this case the effect of charge is eliminated by binding a negatively charged detergent, SDS, to all the proteins which are denatured. The SDS binds uniformly per unit length of protein and therefore the force on the molecules from the field will be a uniform amount per unit length and the only affect on the speed of travel will be the retarding force due to their size. This is therefore a method to separate molecules based on their molecular weights. Clearly not useful for oligomers since these will be forced apart by the SDS.
