Amino Acids, Peptides, and Proteins

Outline

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 (a-amino acids) Two functional groups:

carboxylic acid group amino group on the alpha (a) 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

H3N

CH H

OHH3O
+

OHH3N CH H C O H2N CH H C O

H3O

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]
[zwitterion] = [anion]

pK2

First equivalence point

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

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 O O

pK1
H3N CH C OH H3N CH C O

pK2
H2N CH C O

pKR
H2N CH C O

CH2CH2CH2CH2NH3+

CH2CH2CH2CH2NH3+

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?

O H3N CH CH2 OH C O H3N CH CH2 OH O C OH H2N CH CH2 OH O C O

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 = ?

O heat O OH + H2N R R C NH R + H2O

Structure of amino acid

R H2N C H CO2H

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

O C N H N H O

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)

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

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

Glutamate (Glu/E) vs. Glutamine (Gln/Q) Aspartate (Asp/D) vs. Asparagine (Asn/N) Converted via hydrolysis Use generic abbreviations for either

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 H S CH2 CH2 O

CH C N CH C O

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 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

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 l = 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

N-terminus
+
H2N CH R SO2 Cl

O C

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

H3O+ HCl + + 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

O H C O OH

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

PRIMARY STRUCTURE
The sequence of amino acids
MIL1 sequence: >gi|7662506|ref|NP_056182.1| MIL1 protein [Homo sapiens] MEDCLAHLGEKVSQELKEPLHKALQMLLSQPVTYQAFRECTLETTVHASGWNKILVPLVLLRQML LELTRLGQEPLSALLQFGVTYLEDYSAEYIIQQGGWGTVFSLESEEEEYPGITAEDSNDIYILPS DNSGQVSPPESPTVTTSWQSESLPVSLSASQSWHTESLPVSLGPESWQQIAMDPEEVKSLDSNGA GEKSENNSSNSDIVHVEKEEVPEGMEEAAVASVVLPARELQEALPEAPAPLLPHITATSLLGTRE PDTEVITVEKSSPATSLFVELDEEEVKAATTEPTEVEEVVPALEPTETLLSEKEINAREESLVEE LSPASEKKPVPPSEGKSRLSPAGEMKPMPLSEGKSILLFGGAAAVAILAVAIGVALALRKK length: 386amino acids
Anne-Marie Ternes

PRIMARY STRUCTURE

The numbers of amino acids vary (e.g. insulin 51, lysozyme 129, haemoglobin 574, gamma globulin 1250) The primary structure determines the folding of the polypeptide to give a functional protein Polar amino acids (acidic, basic and neutral) are hydrophilic and tend to be placed on the outside of the protein. Non-polar (hydrophobic) amino acids tend to be placed on the inside of the protein

2007 Paul Billiet ODWS

Infinite variety
The number of possible sequences is infinite An average protein has 300 amino acids, At each position there could be one of 20 different amino acids = 10390 possible combinations Most are useless Natural selection picks out the best

2007 Paul Billiet ODWS

SECONDARY STRUCTURE
The folding of the N-CC backbone of the polypeptide chain using weak hydrogen bonds

Text 2007 Paul Billiet ODWS Science Student

SECONDARY STRUCTURE

This produces the alpha helix and beta pleating The length of the helix or pleat is determined by certain amino acids that will not participate in these structures (e.g. proline)

Text2007 Paul Billiet ODWS

Dr Gary Kaiser

TERTIARY STRUCTURE
The folding of the polypeptide into domains whose chemical properties are determined by the amino acids in the chain

MIL1 protein

2007 Paul Billiet ODWS Anne-Marie Ternes

TERTIARY STRUCTURE

This folding is sometimes held together by strong covalent bonds (e.g. cysteine-cysteine disulphide bridge) Bending of the chain takes place at certain amino acids (e.g. proline) Hydrophobic amino acids tend to arrange themselves inside the molecule Hydrophilic amino acids arrange themselves on the outside

2007 Paul Billiet ODWS

Chain B of Protein Kinase C

Max Planck Institute for Molecular Genetics

When the polypeptide folds into a threedimensional shape, it is called a protein

The three-dimensional shape of a protein is called its tertiary structure

Myoglobin
Binds oxygen Found in the muscles Acts as a storage site for oxygen Makes up the dark meat in chicken

QUATERNARY STRUCTURE
Some proteins are made of several polypeptide subunits (e.g. haemoglobin has four)

Protein Kinase C
Max Planck Institute for Molecular Genetics

Text 2007 Paul Billiet ODWS

QUATERNARY STRUCTURE
These subunits fit together to form the functional protein Therefore, the sequence of the amino acids in the primary structure will influence the protein's structure at two, three or more levels

2007 Paul Billiet ODWS

Shape of the protein is important for its function Ex. Insulin = 51 amino acids

Shape of the protein is important for its function Ex. Insulin = 51 amino acids

Result
Protein structure depends upon the amino acid sequence This, in turn, depends upon the sequence of bases in the gene

2007 Paul Billiet ODWS

PROTEIN FUNCTIONS
Protein structure determines protein function Denaturation or inhibition which may change protein structure will change its function Coenzymes and cofactors in general may enhance the protein's structure

2007 Paul Billiet ODWS

Types of Proteins
Type
Communication Defense Structure Storage Contractile Transport Hormones Enzymes

Function
Cell signaling
Ex. Hormones in the bloodstream

Protection from infection

Ex. Antibodies in the bloodstream

Mechanical support
Ex. Collagen in skin & keratin in hair/nails

Stores nutrients
Ex. Albumin in egg whites

Movement
Ex. Actin and myosin in muscles

Carries other molecules

Ex. Hemoglobin

Chemical messengers
Ex. Growth hormone stimulates bone growth

Speed up chemical reactions Ex. Catalase

Fibrous proteins
Involved in structure: tendons ligaments blood clots (e.g. collagen and keratin) Contractile proteins in movement: muscle, microtubules (cytoskelton, mitotic spindle, cilia, flagella)

2007 Paul Billiet ODWS

Just for fun facts:

Your hair is composed of all a-helix Spider webs are all -pleated sheets

Globular proteins
most proteins which move around (e.g. albumen, casein in milk) Proteins with binding sites: enzymes, haemoglobin, immunoglobulins, membrane receptor sites

2007 Paul Billiet ODWS

Antibodies are Produced by B Lymphocytes

Antibodies are Proteins that Recognize Specific Antigens

Epitopes: Antigen Regions that Interact with Antibodies