AminoAcids Proteins

University of Balamand
Faculty of Medicine & Medical Sciences
Medical Biochemistry & Nutrition

Amino Acids & Proteins J. Attieh
Jihad Attieh, Ph.D.

Faculty of Sciences
Dean
Murr 243 jihad.attieh@balamand.edu.lb
3779
Topics To Be Covered
Lecture One: Amino Acids: Structure & Properties Proteins: Three-Dimensional Structure
Lecture Two: Handling Proteins
Amino Acids & Proteins
J. Attieh
Amino Acids The Building-Blocks of Proteins
J. Attieh
Amino Acids Share a Common Structure
Amino Acids Share a 3-dimensional Tetrahedral Structure

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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
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Aromatic a.a. have characteristic absorption spectra
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Hydroxyl
Sulfhydryl
Amide
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Reactions of Cys: Disulfide Bridges
Also with iodoacetamide, P-mercaptobenzoate & 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellmans reagent)
Imidazole Guanidino
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Note these structural features All 20 a.a. are -amino acids For 19 of the 20, the -amino group is primary; for proline, it is secondary With the exception of glycine, the -carbon of each is a stereocenter Isoleucine and threonine contain a second stereocenter
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Amino Acids Act as Acids & Bases
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Acid-Base Chemistry
Amino Acids are Weak Polyprotic Acids H2A+ + H2O HA0 + H3O+ Ka1 = [ HA0 ] [ H3O+ ] [H2A+ ]
__________________________
Acid-Base Chemistry
The second dissociation (the amino group in the case of glycine): HA0 + H2O A + H3O+ Ka2 = [ A ] [ H3O+ ]
_______________________
[ HA0 ]
Amino Acids Have Characteristic Titration Curves

Diprotic acid
Predominant Form
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Polyprotic Acid
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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: -COOH Groups

In fact, the average pKa of an -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 an electron-withdrawing inductive effect pKa = 2.19 RC HCO O H + H 2 O N H3
+
RC HCO O + H 3 O N H3
+
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Acidity: -NH3+ groups

The average value of pKa for an -NH3+ group is 9.47, compared with a value of 10.76 for an alkylammonium ion
pKa = 9.47
RCHC OO + H 2 O
RCHC OO N H2
N H3
+ H3O
CH 3 CHCH 3 + H 2 O N H3
pKa = 10.76
CH 3 C HCH 3 + H 3 O N H2
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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 -COOH
COOH + H 2 O pKa = 2.00 COO
-
+ H3 O
writing the acid ionization constant and rearranging terms gives

Ka = [H 3O + ] [ -COO - ] [ -COO H] or [ -COO - ] [ -COO H] = Ka [H 3O + ]
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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
[ -COO - ] [ -COO H] = Ka [H 3O ]
+
1.00 x 10-2 1.00 x 10

-7
= 1.00 x 105
at pH 7.0, the -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 [-COO-] to [-COOH] and the net charge on the -carboxyl at that pH
Ionization vs pH
substituting values for Ka of an -NH3+ group and the hydrogen ion concentration at pH 7.0 gives
[ -NH 2 ] [ -NH 3 ]
+
Ka = [H 3 O+ ]
1.00 x 10 -10 1.00 x 10

-7
= 1.00 x 10 -3
at pH 7.0, the ratio of -NH2 to -NH3 + is approximately 1 to 1000 at this pH, an -amino group is 99.9% in the acid or protonated form and has a charge of +1
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
+ pI = 1 2 (p K a COOH + p Ka N H 3 )
= 1 (2.35 + 9.78) = 6.06 2
given in the following tables are isoelectric pH values for the 20 protein-derived amino acids
Isoelectric pH (pI)
Table 3.2 pKa and pI of -amino acids Nonpolar & polar side chains alanine asparagine glutamine glycine isoleucine leucine methionine phenylalanine proline serine threonine tryptophan valine pKa of COOH 2.34 2.02 2.17 2.34 2.36 2.36 2.28 1.83 1.99 2.21 2.63 2.38 2.32 pKa of N H 3 + 9.69 8.80 9.13 9.60 9.68 9.68 9.21 9.13 10.60 9.15 10.43 9.39 9.62 pK a of Side Chain ---------------------------------------pI 6.02 5.41 5.65 5.97 6.02 6.02 5.74 5.48 6.30 5.68 6.53 5.89 5.97
Isoelectric pH (pI)
Table 3.2 (cont'd) Acidic Side Chains aspartic acid glutamic acid cysteine tyrosine Basic Side Chains arginine histidine lysine pKa of COOH 2.10 2.10 2.05 2.20 pKa of COOH 2.01 1.77 2.18 pKa of N H 3 9.82 9.47 10.25 9.11 pKa of N H 3 + 9.04 9.18 8.95
+
pKa of Side Chain 3.86 4.07 8.00 10.07 pKa of Side Chain 12.48 6.10 10.53
pI 2.98 3.08 5.02 5.63
pI 10.76 7.64 9.74
From Amino Acids to Proteins
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Amino Acids Can Establish Peptide Bonds
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Proteins are Linear Polymers of Amino Acids
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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 Bond
Six atoms of the peptide group lie in a plane!
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Proteins: The Three Dimensional Organization OR Secondary, Tertiary, and Quaternary Structure
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Architecture of Protein Molecules

Levels of Protein Structure
Primary, Secondary, Tertiary & Quaternary
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Architecture of Protein Molecules

Fibrous, Globular, Membrane
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Many proteins contain chemical groups other than a.a.
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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
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Secondary Structure
The 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
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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
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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
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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
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Classes of Secondary Structure

All 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
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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
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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
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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
The Alpha Helix

coil of the helix is clockwise or righthanded 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
J. Attieh
Several factors can disrupt an -helix

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

Composed 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
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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
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Supersecondary structures
The combination of - and -sections: unit: two parallel strands of -sheet connected by a stretch of -helix unit: two antiparallel -helices Greek key (Omega): a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself -barrel: created when -sheets are extensive enough to fold back on themselves
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Tertiary Structure
Several important principles: Tertiary structures form wherever possible (due to formation of large numbers of H-bonds) Helices and sheets often pack close together
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Tertiary Structure
Several 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
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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 Proteins
Some 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 alphahelix and beta-sheet Packing of residues is close Empty spaces exist & are in the form of small cavities
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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 C
Phosphoglycerate kinase Domain 2
Phosphorylase Domain 2 Amino Acids & Proteins
Triose phosphate isomerase
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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
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Flavodoxin Triose phosphate isomerase (top)
Influenza Virus Hemagglutinin HA2
Streptomyces subtilisin inhibitor
Ferredoxin
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Protein Folding Molecular 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)
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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 37C Entropy loss due to association unfavorable. Entropy gain due to burying of hydrophobic groups - very favorable!
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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
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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
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Denaturation can be brought about by: 9heat 9large changes in pH, which alter charges on side chains, e.g., -COO- to -COOH or -NH3+ to -NH2 9detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions 9urea or guanidine, which disrupt hydrogen bonding 9mercaptoethanol, which reduces disulfide bonds

AminoAcids Proteins

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University of Balamand

Faculty of Medicine & Medical Sciences

Medical Biochemistry & Nutrition

Jihad Attieh, Ph.D.

Murr 243 jihad.attieh@balamand.edu.lb

Lecture Two: Handling Proteins

Amino Acids & Proteins

Amino Acids The Building-Blocks of Proteins

Amino Acids & Proteins

Amino Acids Share a Common Structure

Amino Acids Share a 3-dimensional Tetrahedral Structure

Amino Acids & Proteins

20 Common Amino Acids

Amino Acids & Proteins

Amino Acids & Proteins

Amino Acids & Proteins

Aromatic a.a. have characteristic absorption spectra

Amino Acids & Proteins

Amino Acids & Proteins

Reactions of Cys: Disulfide Bridges

Amino Acids & Proteins

Amino Acids & Proteins

Amino Acids Act as Acids & Bases

Amino Acids & Proteins

Amino Acids Have Characteristic Titration Curves

Amino Acids & Proteins

Amino Acids & Proteins

pKa Values of the Amino Acids

Acidity: -COOH Groups

Acidity: -NH3+ groups

writing the acid ionization constant and rearranging terms gives

Amino Acids & Proteins

1.00 x 10-2 1.00 x 10

1.00 x 10 -10 1.00 x 10

= 1 (2.35 + 9.78) = 6.06 2

pI 2.98 3.08 5.02 5.63

pI 10.76 7.64 9.74

From Amino Acids to Proteins

Amino Acids & Proteins

Amino Acids Can Establish Peptide Bonds

Amino Acids & Proteins

Proteins are Linear Polymers of Amino Acids

Amino Acids & Proteins

The Peptide Bond

The Coplanar Nature of the Peptide Bond

Six atoms of the peptide group lie in a plane!

Amino Acids & Proteins

Amino Acids & Proteins

Amino Acids & Proteins

Amino Acids & Proteins

Amino Acids & Proteins

Architecture of Protein Molecules

Amino Acids & Proteins

Architecture of Protein Molecules

Amino Acids & Proteins

Many proteins contain chemical groups other than a.a.

Amino Acids & Proteins

How do proteins recognize and interpret the folding information?

Amino Acids & Proteins

Consequences of the Amide Plane

The angles phi and psi are shown here

Amino Acids & Proteins

Steric Constraints on phi & psi