aminoacids proteins

J. AttiehAmino Acids & Proteins

University of Balamand

Faculty of Medicine&

Medical Sciences

Medical Biochemistry& Nutrition


Faculty of SciencesDean

3779

[email protected]

Jihad Attieh, Ph.D.

Murr 243


Topics To Be Covered

Lecture One:• Amino Acids: Structure & Properties• Proteins: Three-Dimensional Structure

Lecture Two:• Handling Proteins


Amino AcidsThe Building-Blocks

of Proteins


Amino Acids Share a Common Structure

Amino Acids Share a 3-dimensional Tetrahedral Structure



20 Common Amino Acids

Classified in four major groups based on side-chain chemistry

• Non-polar amino acids• Polar, uncharged amino acids• Basic (positively charged) amino acids• Acidic (negatively charged) amino acids




Aromatic a.a. have characteristic absorption spectra


Amide

Hydroxyl Sulfhydryl


Reactions of Cys: Disulfide Bridges

Also with iodoacetamide, P-mercaptobenzoate& 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent)


Guanidino

Imidazole



Note these structural features

All 20 a.a. are α-amino acidsFor 19 of the 20, the α-amino group is primary; for proline, it is secondaryWith the exception of glycine, the α-carbon of each is a stereocenterIsoleucine and threonine contain a second stereocenter


Amino Acids Act as Acids & Bases


Acid-Base Chemistry

Amino Acids are Weak Polyprotic Acids

• H2A+ + H2O → HA0 + H3O+

• Ka1 = [ HA0 ] [ H3O+ ] __________________________

[H2A+ ]


Acid-Base ChemistryThe second dissociation

(the amino group in the case of glycine):

• HA0 + H2O → A¯ + H3O+

• Ka2 = [ A¯ ] [ H3O+ ] _______________________

[ HA0 ]


Diprotic acid

Predominant Form

Amino Acids Have Characteristic Titration Curves


Polyprotic Acid


pKa Values of the Amino Acids

• Alpha carboxyl group - pKa = 2• Alpha amino group - pKa = 9• These numbers are approximate, but

significantly different from regular values for such groups in non-amino acid setups.


Acidity: α-COOH Groups In fact, the average pKa of an α-carboxyl group

is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76)

the greater acidity of the amino acid carboxyl group is due to the electron-withdrawing inductive effect of the

-NH3+ group

The ammonium ion has anelectron-withdrawing inductive effect

+pKa = 2.19

NH3+NH3

+RCHCOO-RCHCOOH H3O+H2O+


Acidity: α-NH3+ groups

The average value of pKa for an α-NH3+ group is

9.47, compared with a value of 10.76 for an alkylammonium ion

+pKa = 9.47

NH3+ NH2

RCHCOO- RCHCOO-+ H2O H3O+

pKa = 10.76

NH3+ NH2

CH3CHCH3 CH3CHCH3 + H3O++ H2O


Ionization vs pH• Given the value of pKa of each functional

group, we can calculate the ratio of each acid to its conjugate base as a function of pH

• Consider the ionization of an α-COOH

– writing the acid ionization constant and rearranging terms gives

[α-COO H]

[α-COO -]Ka =

[H3O+]=

Ka

[α-COO H]

[α-COO -]

[H3O+]or

pKa = 2.00α−COO-α−COOH + H3O++ H2O


Ionization vs pH– substituting the value of Ka (1 x 10-2) for the

hydrogen ion concentration at pH 7.0 (1.0 x 10-7) gives

– at pH 7.0, the α-carboxyl group is virtually 100% in the ionized or conjugate base form, and has a net charge of -1

– we can repeat this calculation at any pH and determine the ratio of [α-COO-] to [α-COOH] and the net charge on the α-carboxyl at that pH

=Ka

[α-COO H]

[α-COO -]

[H3O+]= 1.00 x 105

1.00 x 10-7

1.00 x 10-2

=


Ionization vs pH

– substituting values for Ka of an α-NH3+ group and

the hydrogen ion concentration at pH 7.0 gives

– at pH 7.0, the ratio of α-NH2 to α-NH3 + is

approximately 1 to 1000– at this pH, an α-amino group is 99.9% in the acid

or protonated form and has a charge of +1

[α-NH 2]

[α-NH 3+]

Ka=[H3O+]

=1.00 x 10-10

1.00 x 10-7= 1.00 x 10-3

Isoelectric pH

• Isoelectric pH, pI: the pH at which the majority of molecules of a compound in solution have no net charge– the pI for glycine, for example, falls midway

between the pKa values for the carboxyl and amino groups

– given in the following tables are isoelectric pH values for the 20 protein-derived amino acids

pI = 12 (pKa α−COOH + pKa α−NH3

+)

= 21 (2.35 + 9.78) = 6.06

Isoelectric pH (pI)

α−NH3+α−COOH

6.025.415.655.976.026.025.745.486.305.686.535.895.97

pKa ofpKa ofpKa ofpI

----------------

------------------------------------

valine 2.32 9.62tryptophan 2.38 9.39

10.432.63threonineserine 2.21 9.15

10.601.99prolinephenylalanine 1.83 9.13

9.212.28methionine9.682.36leucine

isoleucine 2.36 9.68glycine 2.34 9.60

9.132.17glutamine8.802.02asparagine9.692.34alanine

Side Chain

Nonpolar &polar side chains

Table 3.2 pKa and pI of α-amino acids

Isoelectric pH (pI)

α−NH3+α−COOH

α−NH3+α−COOH

10.76

2.98

5.023.08

7.649.74

5.63

pKa ofpKa ofpKa ofpI

10.079.112.20tyrosine

lysine 2.18 8.95 10.536.109.181.77histidine

glutamic acid 2.10 9.47 4.078.0010.252.05cysteine

aspartic acid 2.10 9.82 3.86

arginine 2.01 9.04 12.48

Side ChainAcidicSide Chains

pKa ofpKa ofpKa ofSide Chain

BasicSide Chains pI

Table 3.2 (cont'd)


From Amino Acidsto Proteins


Amino Acids Can Establish Peptide Bonds


Proteins are Linear Polymers of Amino Acids


The Peptide Bond• is usually found in the trans conformation• has partial (40%) double bond character• is about 0.133 nm long - shorter than a

typical single bond but longer than a double bond

• Due to the double bond character, the six atoms of the peptide bond group are always planar!

• N partially positive; O partially negative


The Coplanar Nature of the Peptide BondSix atoms of the peptide group lie in a plane!



Proteins: The Three Dimensional Organization

OR

Secondary, Tertiary, and Quaternary Structure


Levels of Protein StructurePrimary, Secondary, Tertiary & Quaternary

Architecture of Protein Molecules


Architecture of Protein Molecules

• Fibrous, Globular, Membrane


Many proteins contain chemical groupsother than a.a.


Primary Structure

The Role of the Sequence in Protein Structure

All of the information necessary for folding the peptide chain into its "native” structure is contained in the primary amino acid structure of the peptide.


How do proteins recognize and interpret the folding information?

• Certain loci along the chain may act as nucleation points

• Protein chain must respect thermodynamic considerations

• Chaperones considerably help


Secondary StructureThe atoms of the peptide bond lie in a plane

• The resonance stabilization energy of the planar structure is 88 kJ/mol

• A twist about the C-N bond involves a twist energy of 88 kJ/mol times the square of the twist angle.

• Twists can occur about either of the bonds linking the alpha carbon to the other atoms of the peptide backbone


Consequences of the Amide Plane

Two degrees of freedom per residue for the peptide chain

• Angle about the C(alpha)-N bond is denoted phi• Angle about the C(alpha)-C bond is denoted psi• The entire path of the peptide backbone is

known if all phi and psi angles are specified • Some values of phi and psi are more likely than

others.


The angles phi and

psi are shown here


Steric Constraints on phi & psi

Unfavorable orbital overlap precludes some combinations of phi and psi

• phi = 0, psi = 180 is unfavorable • phi = 180, psi = 0 is unfavorable • phi = 0, psi = 0 is unfavorable


Steric Constraints on phi & psi

• G. N. Ramachandran was the first to demonstrate the convenience of plotting phi, psi combinations from known protein structures

• The sterically favorable combinations are the basis for preferred secondary structures



Classes of Secondary StructureAll these are local structures that are stabilized by hydrogen bonds, although many of these exist, two are the major

forms and at the basis of all others:

• Alpha helix • Beta sheet


The Alpha Helix

• First proposed by Linus Pauling and Robert Corey in 1951

• Identified in keratin by Max Perutz • A ubiquitous component of proteins • Stabilized by H-bonds


Hydrogen bonds stabilize the helix structure

The helix can be viewed as a stacked array of peptide planes hinged at the alpha carbons and approximately parallel to the helix


The Alpha Helix

• Residues per turn: 3.6 • Rise per residue: 1.5 Angstroms • Rise per turn (pitch): 3.6 x 1.5A = 5.4

Angstroms • The backbone loop that is closed by any

H-bond in an alpha helix contains 13 atoms

• phi = -60 degrees, psi = -45 degrees


• coil of the helix is clockwise or right-handed

• each peptide bond is s-trans and planar• C=O of each peptide bond is hydrogen

bonded to the N-H of the fourth amino acid away

• C=O----H-N hydrogen bonds are parallel to helical axis

• all R groups point outward from helix

The Alpha Helix



Several factors can disrupt an α-helix

proline creates a bend because of (1) the restricted rotation due to its cyclic structure and (2) its α-amino group has no N-H for hydrogen bondingstrong electrostatic repulsion caused by the proximity of several side chains of like charge, e.g., Lys and Arg or Glu and Aspsteric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr


The Beta-Pleated SheetComposed of beta strands

• Also first postulated by Pauling and Corey, 1951

• There are parallel and antiparallel sheets• Rise per residue:•

– 3.47 Angstroms for antiparallel strands– 3.25 Angstroms for parallel strands– Each strand of a beta sheet may be pictured as

a helix with two residues per turn


• polypeptide chains lie adjacent to one another;• R groups alternate, first above and then below

plane• each peptide bond is trans and planar• C=O and N-H groups of each peptide bond are

perpendicular to axis of the sheet• C=O---H-N hydrogen bonds are between

adjacent sheets and perpendicular to the direction of the sheet

The Beta-Pleated Sheet


The Beta-Pleated Sheet


The Beta Turn(beta bend, tight turn)

• allows the peptide chain to reverse direction

• carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away

• proline and glycine are prevalent in beta turns



Supersecondary structures

The combination of α- and β-sections:

βαβ unit: two parallel strands of β-sheet connected by a stretch of α-helixαα unit: two antiparallel α-helicesGreek key (Omega): a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itselfβ-barrel: created when β-sheets are extensive enough to fold back on themselves


Tertiary StructureSeveral important principles:

• Tertiary structures form wherever possible (due to formation of large numbers of H-bonds)

• Helices and sheets often pack close together


Tertiary StructureSeveral important principles:

• The backbone links between elements of tertiary structure are usually short and direct

• Proteins fold to make the most stable structures (make H-bonds and minimize solvent contact


Fibrous Proteins

• Much or most of the polypeptide chain is organized approximately parallel to a single axis

• Fibrous proteins are often mechanically strong

• Fibrous proteins are usually insoluble • Usually play a structural role in nature


Globular ProteinsSome design principles

• Most polar residues face the outside of the protein and interact with solvent

• Most hydrophobic residues face the interior of the protein and interact with each other

• They tend to be soluble in water and salt solutions

• Nearly all have substantial sections of alpha-helix and beta-sheet

• Packing of residues is close • Empty spaces exist & are in the form of small

cavities


Globular Proteins

The Forces That Drive Folding• Peptide chain must satisfy the constraints

inherent in its own structure • Peptide chain must fold so as to "bury"

the hydrophobic side chains, minimizing their contact with water

• Peptide chains, composed of L-amino acids, have a tendency to undergo a "right-handed twist"


A New Way to Look at Globular Proteins

Look for "layer structures"• Helices and sheets often pack in layers • Hydrophobic residues are sandwiched

between the layers • Outside layers are covered with mostly

polar residues that interact favorably with solvent


Cytochrome CPhosphoglycerate kinaseDomain 2

PhosphorylaseDomain 2

Triose phosphate isomerase


Classes of Globular Proteins

Jane Richardson's classification• Antiparallel alpha helix proteins • Parallel or mixed beta sheet proteins • Antiparallel beta sheet proteins • Metal- and disulfide-rich proteins


Influenza Virus Hemagglutinin HA2

Streptomyces subtilisin inhibitor

Triose phosphate isomerase (top)

Flavodoxin

Ferredoxin


Protein FoldingMolecular Chaperones

• Why are chaperones needed if the information for folding is inherent in the sequence? – to protect nascent proteins from the

concentrated protein matrix in the cell and perhaps to accelerate slow steps

• Chaperone proteins were first identified as "heat-shock proteins" (hsp60 and hsp70)



Quaternary Structure

What are the forces driving quaternary association?

• Typical Kd for two subunits: 10-8 to 10-16M! • These values correspond to energies of 50-100

kJ/mol at 37°C • Entropy loss due to association – unfavorable.• Entropy gain due to burying of hydrophobic

groups - very favorable!


What are the structural and functional advantages driving quaternary

association?Know these!

• Stability: reduction of surface to volume ratio • Genetic economy and efficiency • Bringing catalytic sites together • Cooperativity


Denaturation

The loss of the structural order (secondary, tertiary, quaternary or a combination of these) that gives a protein its biological activity;

that is, the loss of biological activity


Denaturation can be brought about by:

heatlarge changes in pH, which alter charges on side chains, e.g., -COO- to -COOH or -NH3

+ to -NH2

detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactionsurea or guanidine, which disrupt hydrogen bondingmercaptoethanol, which reduces disulfide bonds

aminoacids proteins

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