Chapter 3: Amino Acids, Peptides, and Proteins

Dr. Clower Chem 4202

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

Amino Acids

The building blocks of proteins Also used as single molecules in biochemical pathways 20 standard amino acids (-amino acids) Two functional groups:

carboxylic acid group amino group on the alpha () carbon Properties dictate behavior of AAs
R side chain | H2N C COOH | H

Have different side groups (R)

Zwitterions

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

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

Basic

R group = carboxylic acid Donates H+ Negatively charged

R group = amine Accepts H+ Positively charged His ionizes at pH 6.0

Acid-base Properties

Remember H3PO4 (multiple pKas) AAs also have multiple pKas due to multiple ionizable groups 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:
O O OH O O

H3N

CH H

OHH3O
+

H3N

CH H

OHH3O
+

H2N

CH H

Glycine ion at acidic pH (charge = 1+)

Zwitterion of glycine (charge = 0)

Glycine ion at basic pH (charge = 1-)

Note that the uncharged species never forms

Titration of Glycine

pK1

[cation] = [zwitterion]

pK2

[zwitterion] = [anion] Zwitterion Molecule has no net charge pH = pI (Isoelectric point) pI = average of pKas = (pK1 + pK2) pIglycine = (2.34 + 9.60) = 5.97

First equivalence point

Animation

pI of Lysine

For AAs with 3 pKas, pI = average of two relevant pKa values Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):
O O OH O O CH C

H3N

pK1

pK2
H3N CH C O H2N CH C O CH2CH2CH2CH2NH3+ CH2CH2CH2CH2NH3+

pKR
H2N CH C O

CH2CH2CH2CH2NH3+

CH2CH2CH2CH2NH2

Which species is the isoelectric form? So, pI = (pK2 + pKR) = (8.95 + 10.53) = 9.74

Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)

Learning Check

Would the following ions of serine exist at a pH above, below, or at pI?

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 AAs found in proteins are L geometry

S enantiomer for all except cysteine

D-AAs 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

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

Rigid restricted rotation

Amide linkage is planar, NH and CO are anti

Polypeptides
Linear polymers (no branches) AA monomers linked head to tail Terminal residues:

Free amino group (N-terminus)

on left on right

Free carboxylate group (C-terminus)

pKa values of AAs in polypeptides differ slightly from pKa values of free AAs

Naming Peptides

Name from the free amine (NH3+) Use -yl endings for the names of the amino acids The last amino acid with the free carboxyl group (COO-) uses its amino acid name

(GA)

Amino Acid Ambiguity

Glx/Z Asx/B

X = undetermined or nonstandard AA

Learning Check
Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.
CH3 CH3 CH CH3 CH3 O H3N CH C N H CH O CH C N H SH CH2 O CH C N H S CH2 CH2 O
CH C O

Learning Check

Draw the structural formula of each of the following peptides.

Outline (part II)

Sections 3.3 and 3.4 Separation and purification Protein sequencing

Analysis of primary structure

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 May affect structure (e.g. proteases/peptidase) Control denaturation (0-4C) Control activity of enzymes Reactive Add protecting group to prevent formation of new disulfide bonds Denature or oxidize Store under N2 or Ar Keep concentration high

Presence of enzymes

Temperature

Thiol groups

Exposure to air, water

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 Solubility is temperature dependent

Temperature

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 Anion groups (carboxylates) bind cations

Cation exchangers

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 O

N-terminus
+
H2N CH R SO2 Cl

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

HCl

H3O+ + other free AAs

SO2 HN CH R

O C

SO2 HN CH R

O C OH

Dabsyl chloride and FDNB

Same result as dansyl chloride Dabsyl chloride 1-Fluoro-2,4dinitrobenzene (FDNB)

N N

O S O Cl

Sanger method

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

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 Identify using chromatography Quantify using absorbance or fluorescence Doesnt give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results

After cleavage:

Disadvantages

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