AMINO ACIDS AND PROTEIN

M. Saifur R., Ph.D

 History
Asparagine (N) the one that the fisrst time found (1806). The last of 20 amino acids is threonin (1938). Amino acid has their own trivial name. Usually come from where they are isolated first time. Example :
Asparagine Asparagus Glutamate wheat gluten Tyrosin cheese (Greek: tyros) Glycine sweet (Greek: glycos)

Amino Acids

Two convention for carbon atom numbering: Greek lettering and numbering Greek lettering : a, b, g, d, e, etc. a is start form the carbon atom which is bounded with four diverent atom. Numbering : Simply numbered from the one end of the molecule C1 is the carbon atom with the substituent contining atom of highest atomic number . `

Atom carbon numbering

Amino acids

Common structural features of amino acids

Naturally occuring amino acids

(20)

Chirality of amino acids

Ca is a chiral center

Chirality is a structural property of an object. An object is said to be chiral if its mirror image can not be superimposed with itself by the means of rotations. Since they are nonsuperimposable mirror images of each other, the two forms represent a class of stereoisomers called enantiomers. Molecule with a chiral center is optically ctive (rotate plane polarized light).

Optically active

No sample

With sample Dextro (D) clockwise Levo (L) counter clockwise rotation rotation

All nautarlly occuring amino acids are Levo or L.

 Based on the R group

Particularly is their polarity or tendency to interact with water at biological pH (near pH 7). Range of polarity : non polar or hydrophobic to highly polar or hydrphilic.
Non polar, aliphatic R group Non polar, aromatic R groups Polar, uncharged R groups Positively charged (Basic) R groups Negatively charged (Acidic) R groups

Amino acid classification

Tend to burried inside the protein, Stabilize protein by a means of hydrophobic interaction, Glycine is the smallest amino acid, and make no contribution for hydrophobic interaction. Proline is unique amino acids, its prensenting reduces the protein flexibility.

Tyr and Trp are more polar than Phe.

Hydroxyl group

Sulfuhydryl group

Amide group

More hydrophilic and tend to form the hydrogen bond with water molecule.

Uncommon amino acids

Selenocysteine Methyllysine

Amphoteric
Substance which have a dual nature (as an acid and as a base) Act as an acids (proton donor) Act as a base (proton acceptor)

Zwitter ion

 Chemical point of view: All amino acids are polyprotic acids (contains more than one ionizable groups). The ionizable group is not strongly dissociating ones, The degree of dissociation is depend on pH of medium, All amino acids at laest contains two dissociable hydrogens.

Acid-base chemistry of amino acids

Ionic form of amino acids

pI : the pH at which the the protein have a net charge of zero
O O CH ONH3+ O C NH3+ OO C NH2

NH3+ pH = 2 Cationic form

NH3+ pH = 7 Zwitter ionic

NH2 pH = 12 Anionic form

Depend on the number of charged amino acid residues

pKa of amino acids

Ka = dissociation constant. Back to the definition of acid-base Brnsted-Lowry

Acids Base

Proton Donor Proton Acceptor

Conjugate base Conjugate acid

(1)

..................... (2) ...

......................... (3) ....

What is the pKa and pH relationship.....?

The third equation is HandersonHasselbalch equation

Titration curve of amino acids

Acid-base titration involves the gradual addition or removal of protons. Amino acids have weak acid and weak basic character. Low pH transition leads to titration of the carboxylic group (COOH COO(deprotonated). High pH transition leads to titration of the amino group (NH3+ NH2).

From the titration curve of glycine we can derive several important pieces of information:. 1. Give a quantitative measure of the pKa of each of the two ionizing groups: 2.34 for the COOH group and 9.60 for the -NH3 group. 2. The amino acid has two region of buffering region (extending 1 unit at either side of pKas.

 The titration curve also predict the electric charge of amino acids

1 pI ( pK1 pK 2) 2
Only for the amino acids with two titration stages 1. All amino acids with a single -amino group, a single -carboxyl group, and an R group that does not ionize have titration curves resembling that of glycine. 2. Amino acids with an ionizable R group have more complex titration curves, with three stages corresponding to the three possible ionization steps; thus they have three pKa values. The additional stage for the titration of the ionizable R group merges to some extent with the other two.

The pKR is emerge in between the two other pKa. The isoelectric points reflect the nature of the ionizing R groups present.

Determination of amino acids

Method : Chromatography
Mikhail Tswett, 1903 , use thie technique for sparating the plant pigment (carotenes and chlorophyl)

Mikhail Tswett experiment

IEX Chromatography

A. Mobile phase has low salt concentration B. Loading of sample in low salt concentration of mobile phase therefore the sample can displace the binding of mobile phase. C. Elution phase with the increasing of salt ion on the mobile phase D. Weakly charge of bounding sample is no longer bound onto the staionary phase and are eluted from column. E. Further increasing of salt ion concentrarion of mobile phase therefore elute the higher charged sample. F. The highly charged particle sample are no longer bound.

The type of IEX Chromatography

+ + + + + + + +

+
+ +

Anion exchanger with exchangeable counter ions

Cation exchanger with exchangeable counter ions

Positively charged of stationary phase

Negatively charged of stationary phase

Stationary phase material of IEX Chromatography

Separation of Amino Acids by Ion Exchange Chromatography Sulfonic acid cation exchanger
CH2 CH CH2 CH

SO3-

SO3-

Positive charges on an amino acid are attracted to the resin by electrostatic forces. The column is run in a buffer of specific pH and ionic strength. Amino acids which are more positively charged at this pH will move through the column more slowly than those which are less positively charged. The effective charge of an amino acid at a specific pH: Dp= pI-pH

Amino acid composition analysis is used to quantify the amount of each amino acid in a protein

1. Hydrolyze peptide bonds: 6M NaOH, 100C, 18-24 h (Cys, Ser, Thr destroyed) or 6M HCl, 100C, 18-24 h (Trp destroyed, Ser and Thr partially destroyed,Asn and Gln deaminated to Asp and Glu)
2. Separate amino acids by ion exchange chromatography. This method separates amino acids by their charge at a particular pH. The conditions which release an amino acid from the column reflect the pI of the amino acid.

Methods for detection of NH2-terminal amino acids

2, 4-dinitroflurobenzene (DNFB)-Sangers reagent

Methods for detection of COOH-terminal amino acids

Chemical Methods: Hydrazine

Enzymatic Methods: Carboxypeptidases

Carboxypeptidases remove carboxy-terminal amino acids Carboxypeptidase A: prefers Phe, Trp, Tyr will not cleave Arg, Lys, Pro Carboxypeptidase B: only cleaves Arg and Lys Neither enzyme will cleave if penultimate residue is Pro

Amino Acid Sequence :Edman Degradation

Peptide and Proteins

Nuclephilie

Leaving group

Peptide bonds

Nucleophilie: Group with electron rich Leaving group: Positive or partly positive group (electrophile)

Formation of peptide bonds by condensation Polypeptides 10,000 MW; Proteins > 10,000 MW

PROTEIN STRUCTURE

Protein synthesis (Movie)

Protein Architecture
Primary structure : a sequence of amino acids Secondary structure : helices, sheets, and turn Tertiary structure : side chains packing in 3D structure Quartenary structure : association of subunits
Occurs when the sequence of amino acids linked by hydrgen bond

Occurs when certain atrraction are present between helices and sheets

The importance of protein structure

Structure (ab)2 (tetramer) Red box: Location of Glu6 on beta chain that mutated to Val)

G6V hydrophilic to hydrophobic Causes polymerization of hemoglobin

Peptide bond rotation

Free rotation around the peptide bond prevented

Conformation flexibility

Backbond (the main chain of peptide bond minus side chain) conformation. Torsion or rotation around the N-Ca bond (f) and CaC1bond()

Peptide bond rotation

Free rotation only occurs arround the f and angle

Beta sheet

Alpha helix
The model was proposed by Pauling and Corey.

They found that alpha helix could be very stable because of intrachain hydrogen bond formation.

Secondary structure formation

Right handed ahelix By formation of hydrogen bond between the N-H (proton donor) group and C=O group. Hydrogen bond is formed every i+ 4

Role of hydrogen bond

The structural stability of alpha helices and beta sheet is the result of hydrogen bonding between the amide proton (the hydrogen atom bonded to the nitrogen of the peptide bond) and the carbonyl oxygen (the oxygen atom bonded to the carbon atom in the amide plane).

Electronegativity

Oxygen is slightly negative while hydrogen is slightly positive

Shared electron

Electron cloud

 Hydrogen bonds energies may seem small on the order of 4 to 25 kJ/mole.

Typical hydrogen bond

Tertiary and quarternary structure

NLAFALSELDRITAQ LKLPRHVEEEAARL YREAVRKGLIRGRS IESVMAACVYAACR LLKVPRTLDEIADIA RVDKKEIGRSYRFI ARNLNLTPKKLF

Fold

Determine

Sequence

Structure*)

*) PCNA = Proliferating Cell Nuclear Antigen

Protein folding (why and how...?)

The energy landscape of protein folding

Protein folding (movie)