Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition Chapter 2 Protein Composition and Structure

Functions of some Proteins Catalysis (enzymes) Structural (collagen) Contractile (muscle) Transport (hemoglobin) Storage (myoglobin) Electron transport (cytochromes) Hormones (insulin) Growth factor (EGF) DNA binding (histones) Ribosomal proteins Toxins and venoms (cholera & melittin) Vision (opsins) Immunoglobins

Replication protein around DNA

ConformationaI change Lactoferrin

Levels of Protein Structure Primary structure (1 o ) – sequence of amino acids starting from the N-terminus of the peptide. Secondary structure (2 o ) – conformations of the peptide chain from rotation about the  -Cs, e.g.  -helices and  -sheets, etc. Tertiary structure (3 o ) – three dimensional shape of the fully folded polypeptide chain. Quaternary structure (4 o ) - arrangement of two or more protein chains into multisubunit molecule

Levels of Protein Structure Bonding: 1 o = covalent 2 o = H-bond 3 o = covalent & noncovalent 4 o = noncovalent

Amino Acids The 20 common are those used in making protein on a ribosome using mRNA and tRNA. These are called  -amino acids since each has a carboxyl group and an amino group attached to an  -carbon atom. They differ by the sidechain or “R” group.  + NH 3 -CH-COOH l R

Amino Acid Classification Classification is made using the structure of the side chain, R. (* = essential) 1. None (hydrogen):Gly 2. Non-polar: Aliphatic:Ala, Val*, Leu*, Ile*, Met* Alicyclic:Pro 3. Aromatic:Phe*, Tyr, Trp* 4. Polar uncharged:Ser, Thr*, Asn, Gln 5. Thiol:Cys 6.Acidic:Asp, Glu 7. Basic:Lys*, His*, Arg*

ball & stick model wedge Fischer projection

1 o aliphatic pKa = 13.6 Aromatic pKa = 7.05 Cl - chloride

Amino Acid Names & Codes

Other structures

All are Chiral at the  -carbon atom except Gly

D, L Assignments The convention for making D, L assignments is to draw a Fischer projection with the carboxyl at the top and the R at the bottom. + NH 3 to the left = L to the right = D

L stereochemistry = S, for this case

Amino Acid Ionization, pKas Each of the 20 common  -amino acids has two pKa values, for the carboxyl group and the amino group attached to the  -carbon. + NH 3 -CH 2 -COOH + NH 3 -CH 2 -COO - + H + + NH 3 -CH 2 -COO - NH 2 -CH 2 -COO - + H + Seven of the 20 have an ionizable sidechain and therefore have a third pKa value.

Amino Acid pKas  -COOH  - + NH 3 R (sidechain) Gly Ala Val Leu Ile Met Pro Phe Trp Ser Thr Asn Gln Cys Asp Glu Tyr Lys His Arg

3.86 D, 4.25 E 10.0 Peptide

Effect of Change in pH

Formation of a Peptide Two amino acids are joined together to form a peptide (amide) bond with a loss of HOH. After becoming part of a peptide or protein these are called “residues” due to loss of HOH.

Primary Structure (1 o ) The sequence of amino acids (N-term to C-term) in a peptide or protein is its primary structure. A pentapeptide

Convention for writing a peptide Dashes indicate a knowledge of the sequence of amino acid residues. N-term Tyr-Gly-Asp-Phe-Leu C-term Commas indicate that the residue is known but not the sequence. N-term Tyr,Gly,Asp,Phe,Leu C-term Asx indicates that the residue could be either Asp or Asn (The same applies to Glx). N-term Tyr-Gly-Asx-Phe-Leu C-term

Disulfide Bond Formation

Disulfide Bonds in Insulin

Peptide Bond Resonance.. Due to resonance participation of the unshared pair of electrons on N, amides are neutral.

Peptide Bond Planarity 6 atoms are coplanar

Peptide Bond Structures Less crowded in the favored trans arrangement

A prolyl residue Crowded in both cis and trans arrangements

Rotation Sites,  and  The rotational arrangements about  -carbons of a peptide or protein gives its secondary (2 o ) structure.

 View from N-term to  -carbon

 View from  -carbon to C-term

Ramachandran Plot

Secondary Structure (2 o ) The two most common secondary structures are the  -helix and the  -sheet. Each of these 2 o structures have fairly specific  and  angles. All other rotational angles represent “random” secondary structure. Secondary structure is maintained by hydrogen bonding.  -helix by intramolecular H-bonds  -sheet by intermolecular H-bonds

 -helix in a Ramachandran Plot  = -47  = -57

The  -helix, a helix

The  -helix

Hydrogen Bond Contacts

 -helix in a Protein

 -sheet in a Ramachandran Plot  = +135  = -139

A  -sheet strand

Anti-parallel  -sheet

Parallel  -sheet

Anti-parallel  -sheet

 -sheet structures

 -sheet in a Protein

      

 -turn or hairpin turn A turn is 4-5 aa residues. A  -turn (hairpin) is 4 aa residues.

Loops are usually larger than turns. >5 aa residues

 -Keratin, a fibrous protein, forms an  coiled coil Each strand is a modified  -helix, 3.5 residues/turn. Right-handed helices form a left-handed supercoil.

A heptad repeat

Collagen, a triple helix Gly every third residue; Gly-Pro-HPro is frequent. Interstrand H-bond at Gly. No Cys, so, no -S-S-

Collagen sequences

Tertiary Structure (3 o ) The three dimensional folding of a polypeptide is its tertiary structure. Both the  -helix and  -sheet may exist within the tertiary structure. Generally the distribution of amino acid sidechains in a globular protein finds mostly nonpolar residues in the interior of the protein and polar residues on the surface. Tertiary structure is maintained by noncovalent interactions and disulfide bonding.

Myoglobin, a globular protein

Myoglobin Surface Cross-section Blue = chargedYellow = hydrophobic

Porin, a membrane spanning protein

Motifs, (Supersecondary structure) These are combinations of secondary structures observed in different proteins. Examples: Helix-loop-helix Coiled coil Helix bundle   - hairpin  - meander Greek key  - sandwich

Motifs

Domain A domain is a discrete globular area within protein. There are four general types: All  only  -  helices and loops All   only  - sheet & loops or turns Mixed  alternating   cluster of  then cluster of 

Domain Multiple domains exist in the protein below.

Quaternary Structure (4 o ) is an assembly of 3 o structures (two or more subunits). A dimer

Hemoglobin, a tetramer Quaternary structure is maintained by noncovalent interactions

Denaturing Proteins Denaturing agents destroy the protein 3 o structure (causes the protein to unravel). Methods: Heat; Extremes of pH; Detergents; Mechanical agitation; Mercaptoethanol – breaks -S-S- bonds; 6M guanidine HCl or 10M urea: these are chaotropic agents that break up noncovalent interactions.

Denaturing agents

Disulfide oxidation-reduction Oxidized Reduced

Ribonuclease 4 disulfide bonds. -S-S-

Then removing urea and ME permits reoxidation.

A trace of ME allows reduction/oxidation to occur until the low free energy form is found and >98% of activity is restored. Reoxidation reforms –S-S- but not necessarily in the correct place.

Disulfide Bonds in Insulin Reduction and treatment as with ribonuclease gives <1% restored activity.

Sharp Transition suggests an all or nothing effect in denaturing.

Protein Folding Protein folding typically occurs as the protein comes off of the ribosome. It is cooperative process and is driven in part by the hydrophobic effect to reach a low free energy conformation. Folding is assisted by molecular chaperones and protein disufide isomerase.

Protein Folding The cooperative process is such that the initial formation of small elements of structure accelerate subsequent structure development (folding). In folding, the polypeptide chain goes from a high energy, high entropy state to a low energy, low entropy state. This occurs faster in vivo that in vitro. Molecular chaperones serve to prevent most errors in folding and when such occurs the assist in refolding. Protein disufide isomerase preventing errors in –S-S- Formation and in correcting errors that occur.

Modified amino acid residues Modification occurs after a protein is synthesized.

Modified amino acid residues Modification produces fluorescence in jellyfish.

Lysozyme, an enzyme Space filling Ball & stick

Lysozyme Protein backbone

Lysozyme Ribbon diagrams 3 o structure Active site & -S-S-

End of Chapter 2 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition