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Chapter 3: Amino Acids, Peptides, and Proteins
Dr. Rajabi
Outline (part I)
Sections 3.1 and 3.2
Amino Acids
Chemical structure
Acid-base properties
Stereochemistry
Non-standard amino acids
Formation of Peptide Bonds
The building blocks of proteins Also used as single molecules in biochemical
pathways 20 standard amino acids (-amino acids)( 21
amino acid=Selenocysteine) Two functional groups:
carboxylic acid group amino group on the alpha () carbon
Have different side groups (R) Properties dictate behavior of AAs
Amino Acids
R side chain
| H2N— C —COOH
|H
Both the –NH2 and the –COOH groups in an amino acid
undergo ionization in water.
At physiological pH (7.4), a zwitterion forms Both + and – charges
Overall neutral
Amphoteric Amino group is protonated
Carboxyl group is deprotonated
Soluble in polar solvents due to ionic character
Structure of R also influence solubility
Zwitterions
Classification of Amino Acids
Classify by structure of R Nonpolar
Polar
Aromatic
Acidic
Basic
Nonpolar Amino Acids
Hydrophobic, neutral, aliphatic
Polar Amino Acids
Hydrophilic, neutral, typically H-bond
Disulfide Bonds
Formed from oxidation of cysteine residues
Aromatic Amino Acids
Bulky, neutral, polarity depend on R
Acidic and Basic Amino Acids
Acidic R group = carboxylic
acid Donates H+ Negatively charged
Basic R group = amine Accepts H+
Positively charged His ionizes at pH 6.0
Remember H3PO4 (multiple pKa’s)
AAs also have multiple pKa’s due to multiple ionizable
groups
Acid-base Properties
pK1 ~ 2.2(protonated below 2.2)
pK2 ~ 9.4(NH3
+ below 9.4)
pKR
(when applicable)
Table 3-1
Note 3-letter and 1-letter
abbreviations
Amino acid organization chart
pH and Ionization
Consider glycine:
Note that the uncharged species never forms
H3N CH C
H
OH
O
H3N CH C
H
O
O
H2N CH C
H
O
O
OH-
H3O+
OH-
H3O+
Glycine ion at acidic pH
(charge = 1+)
Zwitterion of glycine (charge = 0)
Glycine ion at basic pH
(charge = 1-)
Titration of Glycine
pK1
[cation] = [zwitterion]
pK2
[zwitterion] = [anion]
First equivalence point Zwitterion Molecule has no net charge pH = pI (Isoelectric point)
pI = average of pKa’s = ½ (pK1 + pK2)
pIglycine = ½ (2.34 + 9.60) = 5.97
Animation
pI of Lysine
For AAs with 3 pKa’s, pI = average of two relevant pKa values
Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
Which species is the isoelectric form?
So, pI = ½ (pK2 + pKR)
= ½ (8.95 + 10.53) = 9.74
Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)
H3N CH C
CH2CH2CH2CH2NH3+
OH
O
H3N CH C
CH2CH2CH2CH2NH3+
O
O
H2N CH C
CH2CH2CH2CH2NH3+
O
O
H2N CH C
CH2CH2CH2CH2NH2
O
O
pK1 pK2 pKR
Learning Check
Would the following ions of serine exist at a pH above, below, or at pI?
H3N CH C
CH2
O
O
OH
H3N CH C
CH2
OH
O
OH
H2N CH C
CH2
O
O
OH
A good buffer at ~ pH 6.
pI =
Histidine
Stereochemistry of AAs
All amino acids (except glycine) are optically active
Fischer projections:
D and L Configurations
d = dextrorotatory l = levorotatory D, L = relative to glyceraldehyde
Importance of Stereochemistry
All AA’s found in proteins are L geometry
S enantiomer for all except cysteine
D-AA’s are found in bacteria
Geometry of proteins affects reactivity (e.g
binding of substrates in enzymes)
Thalidomide
Non-standard Amino Acids
AA derivatives Modification of AA after
protein synthesized
Terminal residues or R
groups
Addition of small alkyl
group, hydroxyl, etc.
D-AAs Bacteria
CHEM 2412 Review
Carboxylic acid + amine = ?
Structure of amino acid
R C OH
O
+ H2N R R C NH
O
+ H2Oheat
R
H2N C CO2H
H
R
The Peptide Bond
Chain of amino acids = peptide or protein Amino acid residues connected by peptide bonds Residue = AA – H2O
The Peptide Bond
Non-basic and non-acidic in pH 2-12 range due to delocalization of N lone pair
Amide linkage is planar, NH and CO are anti
C
O
N
H
O
N
HRigid
restricted rotation
Polypeptides
Linear polymers (no branches) AA monomers linked head to tail Terminal residues:
Free amino group (N-terminus) Draw on left
Free carboxylate group (C-terminus) Draw on right
pKa values of AAs in polypeptides differ slightly from pKa values of free AAs
Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.
Learning Check
CH CH3
CH3
H3N CH C
O
N
H
CH C
O
N
H
CH C
O
N
H
CH C O-
OCH CH2
CH2
S
CH3
CH2
SH
CH3
Learning Check
Draw the structural formula of each of the following peptides.A. Methionylaspartic acid
B. Alanyltryptophan
C.Methionylglutaminyllysine
D.Histidylglycylglutamylalanine
Outline (part II)
Sections 3.3 and 3.4 Separation and purification Protein sequencing
Analysis of primary structure
Protein structure:There are four levels of protein structure (primary, secondary, tertiary and quaternary) Primary structure: • The primary structure of a protein is its unique sequence of amino acids.
– Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids.
– The precise primary structure of a protein is determined by inherited genetic information.
– At one end is an amino acid with a free amino group the (the N-terminus) and at the other is an amino acid with a free carboxyl group the (the C-terminus).
2- Secondary structure: Results from hydrogen bond
formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:
α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one
β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).
I →I+4
Hydrogen bonding in α-helix: In the α-helix CO of the one amino acid residue forms H-bond with NH of the forth one.
Supersecondary structure or Motifs : occurs by combining secondary structure.
The combination may be: α-helix- turn- α-helix- turn…..etc Or: β-sheet -turn- β-sheet-turn………etc
Or: α-helix- turn- β-sheet-turn- α-helixTurn (or bend): is short segment of polypeptides (3-4 amino acids)
that connects successive secondary structures.e.g. β-turn: is small polypeptide that connects successive strands of β-
sheets.
b. Strong covalent bonds include disulfide bridges, that form between the sulfhydryl groups (SH) of cysteine monomers, stabilize the structure.
• Quaternary structure: results from the aggregation (combination) of two or more polypeptide subunits held together by non-covalent interaction like H-bonds, ionic or hydrophobic interactions.
• Examples on protein having quaternary structure:
– Collagen is a fibrous protein of three polypeptides (trimeric) that are supercoiled like a rope.
•This provides the structural strength for their role in connective tissue.
– Hemoglobin is a globular protein with four polypeptide chains (tetrameric)
– Insulin : two polypeptide chains (dimeric)
Summary of Protein Structures
Copyright © 2007 by Pearson Education, Inc Publishing as Benjamin Cummings
Protein size
In general, proteins contain > 40 residues Minimum needed to fold into tertiary structure
Usually 100-1000 residues Percent of each AA varies Proteins separated based on differences in
size and composition Proteins must be pure to analyze, determine
structure/function
Factors to control
pH Keep pH stable to avoid denaturation or chemical degradation
Presence of enzymes May affect structure (e.g. proteases/peptidase)
Temperature Control denaturation (0-4°C) Control activity of enzymes
Thiol groups Reactive Add protecting group to prevent formation of new disulfide bonds
Exposure to air, water Denature or oxidize Store under N2 or Ar Keep concentration high
General Separation Procedure
Detect/quantitate protein (assay) Determine a source (tissue) Extract protein
Suspend cell source in buffer Homogenize
Break into fine pieces Cells disrupted Soluble contents mix with buffer Centrifuge to separate soluble and insoluble
Separate protein of interest Based on solubility, size, charge, or binding ability
Solubility
Selectively precipitate protein Manipulate
Concentration of salts Solvent pH Temperature
Concentration of salts
Adding small amount of salt increases [Protein]
Salt shields proteins from each other, less
precipitation from aggregation Salting-in
Salting out Continue to increase [salt] decreases [protein]
Different proteins salt out at different [salt]
Other Solubility Methods
Solvent Similar theory to salting-out Add organic solvent (acetone, ethanol) to interact with
water Decrease solvating power
pH Proteins are least soluble at pI Isoelectric precipitation
Temperature Solubility is temperature dependent
Chromatography
Mobile phase Mixture dissolved in liquid or
solid
Stationary phase Porous solid matrix
Components of mixture
pass through the column
at different rates based on
properties
Types of Chromatography
Paper Stationary phase = filter paper
Same theory as thin layer chromatography (TLC)
Components separate based on polarity
High-performance liquid (HPLC) Stationary phase = small uniform particles, large surface area
Adapt to separate based on polarity, size, etc.
Hydrophobic Interaction Hydrophobic groups on matrix
Attract hydrophobic portions of protein
Types of Chromatography
Ion-exchange Stationary phase =
chemically modified to
include charged groups
Separate based on net
charge of proteins
Anion exchangers Cation groups (protonated
amines) bind anions
Cation exchangers Anion groups (carboxylates)
bind cations
Types of Chromatography
Gel-filtration Size/molecular exclusion
chromatography Stationary phase = gels
with pores of particular size
Molecules separate based on size
Small molecules caught in pores
Large molecules pass through
Types of Chromatography
Affinity Matrix chemically
altered to include a molecule designed to bind a particular protein
Other proteins pass through
UV-Vis Spectroscopy
Absorbance used to
monitor protein
concentrations of each
fraction
= 280 nm Absorbance of aromatic
side groups
Electrophoresis
Migration of ions in an electric field
Electrophoretic mobility (rate of movement) function of
charge, size, voltage, pH
The positively charged proteins move towards the negative
electrode (cathode)
The negatively charged proteins move toward the positive
electrode (anode)
A protein at its pI (neutral) will not migrate in either direction
Variety of supports (gel, paper, starch, solutions)
Protein Sequencing
Determination of primary structure Need to know to determine 3D structure Gives insight into protein function Approach:
Denature protein Break protein into small segments Determine sequences of segments
Animation
End group analysis
Identify number of terminal AAs Number of chains/subunits
Identify specific AA
Dansyl chloride/dabsyl chloride Sanger method (FDNB) Edman degradation (PITC)
Bovine insulin
Dansyl chloride
Reacts with primary amines N-terminus
Yields dansylated polypeptides Dansylated polypeptides
hydrolyzed to liberate the modified dansyl AA
Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)
Useful method when protein fragmented into shorter polypeptides
N
SO2
Cl
+
HN CH
R
C
O
N
SO2
H2N CH
R
C
O
HCl +H3O+
HN CH
R
C
O
OH
N
SO2
+ other free AAs
Dabsyl chloride and FDNB
Same result as dansyl chloride
Dabsyl chloride
1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger method
SN
NN O
O
Cl
Edman degradation
Phenylisothiocyanate (PITC) Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC) Treat with TFAA (solvent/catalyst) to cleave N-terminal residue Does not hydrolyze other AAs Treatment with dilute acid makes more stable organic compound
Identify using NMR, HPLC, etc. Sequenator (entire process for proteins < 100 residues)
Fragmenting Proteins
Formation of smaller segments to assist with
sequencing
Process: Cleave protein into specific fragments
Chemically or enzymatically
Break disulfide bonds
Purify fragments
Sequence fragments
Determine order of fragments and disulfide bonds
Cleaving Disulfide Bonds
Oxidize with performic acid
Cys residues form cysteic acid
Acid can oxidize other
residues, so not ideal
H C
O
O OH
Cleaving Disulfide Bonds
Reduce by mercaptans (-SH) 2-Mercaptoethanol
HSCH2CH2OH
Dithiothreitol (DTT)
HSCH2CH(OH)CH(OH)CH2SH
Reform cysteine residues
Oxidize thiol groups with
iodoacetete (ICH2CO2-) to
prevent reformation of disulfide
bonds
Hydrolysis
Cleaves all peptide bonds Achieved by
Enzyme Acid Base
After cleavage: Identify using chromatography Quantify using absorbance or fluorescence
Disadvantages Doesn’t give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results
Enzymatic and Chemical Cleavage
Enzymatic Enzymes used to break
protein into smaller peptides
Endopeptidases Catalyze hydrolysis of
internal peptide bonds
Chemical Chemical reagents used to
break up polypeptides Cyanogen bromide (BrCN)
An example
Another example
A protein is cleaved with cyanogen bromide to yield the following sequences: Arg-Ala-Tyr-Gly-Asn Leu-Phe-Met Asp-Met
The same protein is cleaved with chymotrypsin to yield the following sequences: Met-Arg-Ala-Tyr Asp-Met-Leu-Phe Gly-Asn
What is the sequence of the protein?
Suggested Problems, Chapter 3
1-5, 7, 10-13, 15, 18
2- Secondary structure: Results from hydrogen bond
formation between hydrogen of –NH group of peptide bond and the carbonyl oxygen of another peptide bond. According to H-bonding there are two main forms of secondary structure:
α-helix: It is a spiral structure resulting from hydrogen bonding between one peptide bond and the fourth one
β-sheets: is another form of secondary structure in which two or more polypeptides (or segments of the same peptide chain) are linked together by hydrogen bond between H- of NH- of one chain and carbonyl oxygen of adjacent chain (or segment).