Protein Basics

Maureen Hillenmeyer 02-04-02

Protein function Protein structure

Primary
Amino acids Linkage

Protein conformation framework

Dihedral angles Ramachandran plots

Sequence similarity and variation

Protein Function in Cell

1. Enzymes
Catalyze biological reactions

2. Structural role
Cell wall Cell membrane Cytoplasm

Protein Structure

Protein Structure

Model Molecule: Hemoglobin

Hemoglobin: Background
Protein in red blood cells

Red Blood Cell (Erythrocyte)

Hemoglobin: Background
Protein in red blood cells Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen

Heme Groups in Hemoglobin

Hemoglobin: Background
Protein in red blood cells Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen Picks up oxygen in lungs, releases it in peripheral tissues (e.g. muscles)

Hemoglobin Quaternary Structure

Two alpha subunits and two beta subunits (141 AA per alpha, 146 AA per beta)

Hemoglobin Tertiary Structure

One beta subunit (8 alpha helices)

Hemoglobin Secondary Structure

alpha helix

Structure Stabilizing Interactions

Noncovalent
Van der Waals forces (transient, weak electrical attraction of one atom for another) Hydrophobic (clustering of nonpolar groups) Hydrogen bonding

Hydrogen Bonding
Involves three atoms:
Donor electronegative atom (D)
(Nitrogen or Oxygen in proteins)

Hydrogen bound to donor (H) Acceptor electronegative atom (A) in close proximity

DH

D-H Interaction
Polarization due to electron withdrawal from
the hydrogen to D giving D partial negative charge and the H a partial positive charge Proximity of the Acceptor A causes further charge separation - + -

DH

D-H Interaction
Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge Proximity of the Acceptor A causes further charge separation

Result:

DH

Closer approach of A to H Higher interaction energy than a simple van der Waals interaction

Hydrogen Bonding And Secondary Structure

alpha-helix

beta-sheet

Structure Stabilizing Interactions

Noncovalent
Van der Waals forces (transient, weak electrical attraction of one atom for another) Hydrophobic (clustering of nonpolar groups) Hydrogen bonding

Covalent
Disulfide bonds

Disulfide Bonds
Side chain of cysteine contains highly reactive thiol group

Two thiol groups form a disulfide bond

Disulfide Bridge

Disulfide Bonds
Side chain of cysteine contains highly reactive thiol group

Two thiol groups form a disulfide bond Contribute to the stability of the folded state by linking distant parts of the polypeptide chain

Disulfide Bridge Linking Distant Amino Acids

Hemoglobin Primary Structure

NH2-Val-His-Leu-Thr-Pro-Glu-Glu-

Lys-Ser-Ala-Val-Thr-Ala-Leu-TrpGly-Lys-Val-Asn-Val-Asp-Glu-ValGly-Gly-Glu-..
beta subunit amino acid sequence

Protein Structure - Primary

Protein: chain of amino acids joined by peptide bonds

Protein Structure - Primary

Protein: chain of amino acids joined by peptide bonds Amino Acid
Central carbon (C) attached to:
Hydrogen (H) Amino group (-NH2) Carboxyl group (-COOH) Side chain (R)

General Amino Acid Structure

H H2N
C

COOH

General Amino Acid Structure At pH 7.0

H +H3N
C

COO-

General Amino Acid Structure

Amino Acids
Chiral

Chirality: Glyceraldehyde

D-glyderaldehyde

L-glyderaldehyde

Amino Acids
Chiral 20 naturally occuring; distinguishing side chain

20 Naturally-occurring Amino Acids

Amino Acids
Chiral 20 naturally occuring; distinguishing side chain Classification:
Non-polar (hydrophobic) Charged polar Uncharged polar

Alanine: Nonpolar

Serine: Uncharged Polar

Aspartic Acid Charged Polar

Glycine Nonpolar (special case)

Peptide Bond
Joins amino acids

Peptide Bond Formation

Peptide Chain

Peptide Bond
Joins amino acids 40% double bond character
Caused by resonance

Peptide bond
Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length

Peptide Bond Lengths

Peptide bond
Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length Double bond disallows rotation

Protein Conformation Framework

Bond rotation determines protein folding, 3D structure

Bond Rotation Determines Protein Folding

Protein Conformation Framework

Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle)
Measures orientation of four linked atoms in a molecule: A, B, C, D

Protein Conformation Framework

Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle)
Measures orientation of four linked atoms in a molecule: A, B, C, D ABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D

Ethane Rotation

A D B C

D
B C

Protein Conformation Framework

Bond rotation determines protein folding, 3D structure Torsion angle (dihedral angle)
Measures orientation of four linked atoms in a molecule: A, B, C, D ABCD defined as the angle between the normal to the plane of atoms A-B-C and normal to the plane of atoms B-C-D Three repeating torsion angles along protein backbone: , ,

Backbone Torsion Angles

Backbone Torsion Angles

Dihedral angle : rotation about the peptide bond, namely C1-{C-N}- C2

Backbone Torsion Angles

Backbone Torsion Angles

Dihedral angle : rotation about the peptide bond, namely C1-{C-N}- C2 Dihedral angle : rotation about the bond between N and C

Backbone Torsion Angles

Backbone Torsion Angles

Dihedral angle : rotation about the peptide bond, namely C1-{C-N}- C2 Dihedral angle : rotation about the bond between N and C Dihedral angle : rotation about the bond between C and the carbonyl carbon

Backbone Torsion Angles

Backbone Torsion Angles

angle tends to be planar (0 - cis, or 180 trans) due to delocalization of carbonyl electrons and nitrogen lone pair

Backbone Torsion Angles

angle tends to be planar (0 - cis, or 180 trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair and are flexible, therefore rotation occurs here

Backbone Torsion Angles

Backbone Torsion Angles

angle tends to be planar (0 - cis, or 180 trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair and are flexible, therefore rotation occurs here However, and of a given amino acid residue are limited due to steric hindrance

Steric Hindrance
Interference to rotation caused by spatial arrangement of atoms within molecule Atoms cannot overlap Atom size defined by van der Waals radii Electron clouds repel each other

Backbone Torsion Angles

angle tends to be planar (0 - cis, or 180 trans) due to delocalization of carbonyl pi electrons and nitrogen lone pair and are flexible, therefore rotation occurs here However, and of a given amino acid residue are limited due to steric hindrance Only 10% of the {, } combinations are generally observed for proteins First noticed by G.N. Ramachandran

G.N. Ramachandran
Used computer models of small polypeptides to systematically vary and with the objective of finding stable conformations For each conformation, the structure was examined for close contacts between atoms Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii Therefore, and angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone

Ramachandran Plot
Plot of vs.
The computed angles which are sterically allowed fall on certain regions of plot

Computed Ramachandran Plot

White = sterically disallowed conformations (atoms come closer than sum of van der Waals radii) Blue = sterically allowed conformations

Ramachandran Plot
Plot of vs.
Computed sterically allowed angles fall on certain regions of plot Experimentally determined angles fall on same regions

Experimental Ramachandran Plot

, distribution in 42 high-resolution protein structures (x-ray crystallography)

Ramachandran Plot And Secondary Structure

Repeating values of and along the chain result in regular structure For example, repeating values of ~ -57 and ~ -47 give a right-handed helical fold (the alphahelix)

The structure of cytochrome C shows many segments of helix and the Ramachandran plot shows a tight grouping of , angles near -50,-50

alpha-helix

cytochrome C Ramachandran plot

Similarly, repetitive values in the region of = -110 to 140 and = +110 to +135 give beta sheets. The structure of plastocyanin is composed mostly of beta sheets; the Ramachandran plot shows values in the 110, +130 region:

beta-sheet

plastocyanin Ramachandran plot

Ramachandran Plot And Secondary Structure

White = sterically disallowed conformations Red = sterically allowed regions if strict (greater) radii are used (namely righthanded alpha helix and beta sheet) Yellow = sterically allowed if shorter radii are used (i.e. atoms allowed closer together; brings out left-handed helix)

Sample Ramachandran Plot

Alanine Ramachandran Plot

Arginine Ramachandran Plot

Glutamine Ramachandran Plot

Glycine Ramachandran Plot

Note more allowed regions due to less steric hindrance - Turns

Proline Ramachandran Plot

Note less allowed regions due to structure rigidity

, and Secondary Structure

Name Structure ------------------- ------- ------- --------------------------------alpha-L 57 47 left-handed alpha helix 3-10 Helix -49 -26 right-handed. helix -57 -80 right-handed. Type II helices -79 150 left-handed helices formed by polyglycine and polyproline. Collagen -51 153 right-handed coil formed of three left handed helicies.

Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality

Homologous Proteins: Enterotoxin and Cholera toxin

Enterotoxin 80% homology

Cholera toxin

Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality Low sequence similarity implies little structural similarity

Nonhomologous Proteins: Cytochrome and Barstar

Cytochrome

Barstar

Less than 20% homology

Sequence Similarity
Sequence similarity implies structural, functional, and evolutionary commonality Low sequence similarity implies little structural similarity Small mutations generally well-tolerated by native structure with exceptions!

Sequence Similarity Exception

Sickle-cell anemia resulting from one residue change in hemoglobin protein Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine

Sickle-cell mutation in hemoglobin sequence

Normal Trait
Hemoglobin molecules exist as single, isolated units in RBC, whether oxygen bound or not Cells maintain basic disc shape, whether transporting oxygen or not

Sickle-cell Trait
Oxy-hemoglobin is isolated, but deoxyhemoglobin sticks together in polymers, distorting RBC Some cells take on sickle shape

Sickle-cell

RBC Distortion
Hydrophobic valine replaces hydrophilic glutamate Causes hemoglobin molecules to repel water and be attracted to one another Leads to the formation of long hemoglobin filaments

Hemoglobin Polymerization

Normal

Mutant

RBC Distortion
Hydrophobic valine replaces hydrophilic glutamate Causes hemoglobin molecules to repel water and be attracted to one another Leads to the formation of long hemoglobin filaments Filaments distort the shape of red blood cells (analogy: icicle in a water balloon) Rigid structure of sickle cells blocks capillaries and prevents red blood cells from delivering oxygen

Capillary Blockage

Sickle-cell Trait
Oxy-hemoglobin is isolated, but deoxyhemoglobin sticks together in polymers, distorting RBC Some cells take on sickle shape When hemoglobin again binds oxygen, again becomes isolated Cyclic alteration damages hemoglobin and ultimately RBC itself

Protein: The Machinery of Life

NH2-Val-His-Leu-Thr-Pro-Glu-GluLys-Ser-Ala-Val-Thr-Ala-Leu-TrpGly-Lys-Val-Asn-Val-Asp-Glu-ValGly-Gly-Glu-..

Life is the mode of existence of proteins, and this mode of existence essentially consists in the constant selfrenewal of the chemical constituents of these substances. Friedrich Engles, 1878