Chapter 1/Structure I

• The Building Blocks

• Chemical Properties of Polypeptide Chains

Level of Protein Structure

The amino acid sequence of a protein's polypeptide chain is called its primary structure. Different regions of the sequence form local regular secondary structures, such as alpha () helices or beta () strands. The tertiary structure is formed by packing such structural elements into one or several compact globular units called domains. The final protein may contain several polypeptide chains arranged in a quaternary structure. By formation of such tertiary and quaternary structures, amino acids far apart in the sequence are brought close together in three dimensions to form a functional region, called an active site.

Amino Acids• Proteins are built up by

amino acids that are linked by peptide bonds to form a polypeptide chain.

• An amino acid has several structural components:– A central carbon atom

(C) is attached to – an amino group (NH2), – a carboxyl group

(COOH), – a hydrogen atom (H),– a side chain (R).

Polypeptide Chain

• In a polypeptide chain the carboxyl group of the amino acid n has formed a peptide bond, C-N, to the amino group of the amino acid n + 1. One water molecule is eliminated in this process. The repeating units, which are called residues, are divided into main-chain atoms and side chains. The main-chain part, which is identical in all residues, contains a central C atom attached to an NH group, a C'=O group, and an H atom. The side chain R, which is different for different residues, is bound to the Catom.

The “Handedness" of Amino Acids.

• Looking down the H-Cbond from the hydrogen atom, the L-form has CO, R, and N substituents from C going in a clockwise direction. For the L-form the groups read CORN in the clockwise direction.

• All a.a. except Gly (R = H) have a chiral center• All a.a. incorporated into proteins by organisms are in the L-form.

Hydrophobic Amino Acids

Charged Amino Acids

Polar Amino Acids

Chemical Structure of Gly

• Glycine

Gly

G

Glycine• Relative abundance

7.5 %

• flexible, seen in turns

Chemical Structure of Ala

• Alanine

Ala

A

Alanine

• Relative abundance 9.0 %

• hydrophobic, unreactive,

-helix former

Chemical Structure of Val

• Valine

Val

V

Valine

• Relative abundance

6.9 %

• hydrophobic, unreactive, stiff,

-substitution

-sheet former

Chemical Structure of Leu

• Leucine

Leu

L

Leucine

• Relative abundance

7.5 %

• hydrophobic, unreactive,

-helix, -sheet former

Chemical Structure of Ile

• Isoleucine

Ile

I

Isoleucine• Relative abundance

4.6%

• hydrophobic, unreactive, stiff,

-substitution

-sheet former

Chemical Structure of Met

• Methionene

Met

M

Methionine

• Relative abundance 1.7 %

• thio-ether,

un-branched nonpolar,

ligand for Cu2+ binding

-helix former

Chemical Structure of Cys

• Cysteine

Cys

C

Cysteine• pKa = 8.33• Relative abundance

2.8 %

• thiol, disulfide cross-links, nucleophile in proteases

ligand for Zn2+ binding

-sheet, -turn former

Disulfide Bonds

• Disulfide bonds form between the side chains of two cysteine residues.

• Two SH groups from cysteine residues, which may be in different parts of the amino acid sequence but adjacent in the three-dimensional structure, are oxidized to form one S-S (disulfide) group.

2 -CH2SH + 1/2 O2 -CH2-S-S-CH2 + H2O

Chemical Structure of Pro

• Proline

Pro

P

Proline• Relative abundance

4.6 %

• 2° amine, stiff,

20 % cis, slow isomerization

seen in turns• Initiation of -helix

Chemical Structure of Phe

• Phenylalanine Phe F Fenylalanine• Relative abundance

3.5 %

• hydrophobic, unreactive, polarizableabsorbance at 257 nm

Chemical Structure of Trp• Tryptophan

Trp

W

tWo rings• Relative abundance

1.1 %

• largest hydrophobic, absorbance at 280 nm fluorescent ~340 nm,

exhibits charge transfer

Chemical Structure of Tyr• Tyrosine

Tyr

Y

tYrosine• pKa = 10.13• Relative abundance

3.5 %

• aromatic,

absorbance at 280 nm

fluorescent at 303 nm• can be phosphorylated

hydroxyl can be nitrated, iodinated, & acetylated

Chemical Structure of Ser• Serine

Ser

S

Serine• Relative abundance

7.1 %

• hydroxyl, polar, H-bonding ability

• nucleophile in serine proteases

phosphorylation and glycosylation

The Catalytic Triad of Trypsin

Chemical Structure of Thr• Threonine

Thr

T

Threonine• Relative abundance

6.0 %

• hydroxyl, polar, H-bonding ability,

stiff,

substitution

phosphorylation and glycosylation

Chemical Structure of Asp• Aspartic Acid

Asp

D

AsparDic• pKa = 3.90• Relative abundance

5.5 %

• carboxylic acid, in active sites forcleavage of C-O bonds,member of catalytic triad in serine proteases acts in general acid/base catalysis, ligand for Ca2+ binding

Calcium-binding Site in Calmodulin

Chemical Structure of Glu• Glutamic Acid

Glu

E

GluEtamic• pKa = 4.07• Relative abundance 6.2

%

• carboxylic acid,

ligand for Ca2+ bindingas acts as a general acid/base in catalysis for lysozyme, proteinase

Chemical Structure of Asn• Asparagine

Asn

N

AsparagiNe• Relative abundance

4.4 %

• Polar,

acts as both H-bond donor and acceptor

molecular recognition site can be hydrolyzed to Asp

Chemical Structure of Gln• Glutamine

Gln

Q

Qutamine• Relative abundance

3.9%

• Polar, acts as both H-bond donor and acceptor

• molecular recognition site can be hydrolyzed to Asp

N-terminal Gln can be cyclized

Chemical Structure of Lys• Lysine

Lys

K

Before L• pKa = 10.79• Relative abundance

7.0 %

• amine base, floppy, charge interacts with phosphate DNA/RNAforms schiff base with aldehydes (-N-N=CH-)

• a catalytic residue in some enzymes

Chemical Structure of Arg• Arginine

ArgR

aRginine• pKa = 12.48• Relative abundance

4.7 %

• Guanidine group,

good charge coupled with acid

charge interacts with phosphate

DNA/RNA

a catalytic residue in some enzymes

Chemical Structure of His• Histidine

His

H

Histidine• pKa = 6.04• Relative abundance

2.1 %

• imidazole acid or base;

pKa = pH (physiological),

member of catalytic triad in serine proteases

ligand for Zn2+ and Fe3+ binding

Properties of the Peptide Bond

• Each peptide unit contains the C atom and the C'=O group of the residue n as well as the NH group and the C atom of the residue n + 1.

• Each such unit is a planar, rigid group with known bond distances and bond angles. R1, R2, and R3 are the side chains attached to the Catoms that link the peptide units in the polypeptide chain.

• The peptide group is planar because the additional electron pair of the C=O bond is delocalized over the peptide group such that rotation around the C-N bond is prevented by an energy barrier.

Resonance Tautomers of a Peptide

Peptide Bond

• The peptide bonds are planer in proteins

and almost always trans.

• Trans isomers of the peptide bond are 4 kcal/mol more stable than cis isomers =>

• 0.1 % cis.

Polypeptide Chain

• Each peptide unit has two degrees of freedom; it can rotate around two bonds, its C-C' bond and its N-C bond.

• The angle of rotation around the N-C bond is called phi () and that around the C-C' bond is called psi ().

• The conformation of the main-chain atoms is determined by the values of these two angles for each amino acid.

Torsion Angles Phi and Psi

Ramachandran Plots

• Ramachandran plots indicate allowedcombinations of the conformational angles phi and psi.

• Since phi () and psi () refer to rotations of two rigid peptideunits around the same C atom, mostcombinations produce stericcollisions either between atoms in different peptide groups orbetween a peptide unit and the side chain attached to C. Thesecombinations are therefore not allowed.

• Colored areas show sterically allowed regions. The areas labeled andLcorrespond approximately to conformational angles found for theusual right-handed helices, strands, and left-handed helices,respectively.

Calculated Ramachandran Plots for Amino Acids

• (Left) Observed values for all residue types except glycine. Each point represents and values for an amino acid residue in a well-refined x-ray structure to high resolution.

• (Right) Observed values for glycine. Notice that the values include combinations of and that are not allowed for other amino acids. (From J. Richardson, Adv. Prot. Chem. 34: 174-175,1981.)

Gly with only one H atom as a sidechain, can adopt a much wider range of conformations thanthe other residues.

Certain Side-chain Conformations are Energetically Favorable

• The staggered conformations are the most energetically favored conformations of two tetrahedrally coordinated carbon atoms.

3 conformations of Val

Side Chain Conformation

• The side chain atoms of amino acids are named using the Greek alphabet according to this scheme.

Side Chain Torsion Angles

• The side chain torsion angles are named chi1, chi2, chi3, etc., as shown below for lysine.

Chi1(χ1) Angles• The chi1 angle is subject to

certain restrictions, which arise from steric hindrance between the gamma side chain atom(s) and the main chain.

• The different conformations of the side chain as a function of chi1 are referred to as gauche(+), trans and gauche(-). These are indicated in the diagrams here, in which the amino acid is viewed along the C-C bond.

The most abundant conformation is gauche(+), in which the gamma side chain atom is opposite to the residue's main chain carbonyl group when viewed along the C-C bond.

Gauche

The second most abundant conformation is trans, in which the side chain gamma atom is opposite the main chain nitrogen.

The least abundant conformation is gauche(-), which occurs when the side chain is opposite the hydrogen substituent on the C atom. This conformation is unstable because the gamma atom is in close contact with the main chain CO and NH groups. The gauche(-) conformation is occasionally adopted by Ser or Thr residues in helices.

Chi2 (2)• In general, side chains tend to adopt the same

three torsion angles (+/- 60 and 180 degrees) about chi2 since these correspond to staggered conformations.

• However, for residues with an sp2 hybridized gamma atom such as Phe, Tyr, etc., chi2 rarely equals 180 degrees because this would involve an eclipsed conformation. For these side chains the chi2 angle is usually close to +/- 90 degrees as this minimizes close contacts.

• For residues such as Asp and Asn the chi2 angles are strongly influenced by the hydrogen bonding capacity of the side chain and its environment. Consequently, these residues adopt a wide range of chi2 angles.

Many Proteins Contain Intrinsic Metal Atoms • (a) The di-iron center of the

enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules.

• (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains.

EF-hand Calcium-binding Motif

• The calcium atom is bound to one of the motifs in the muscle protein troponin-C through six oxygen atoms: one each from the side chains of Asp (D) 9, Asn (N) 11, and Asp (D) 13; one from the main chain of residue 15; and two from the side chain of Glu (E) 20. In addition, a water molecule (W) is bound to the calcium atom.