Paul D. Adams • University of Arkansas

Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell

Chapter FourThe Three-Dimensional Structure of Proteins

Protein Structure

• Many conformations are possible for proteins:• Due to flexibility of amino acids linked by peptide

bonds

• At least one major conformations has biological activity, and hence is considered the protein’s native conformation

Levels of Protein Structure

1° structure: the sequence of amino acids in a polypeptide chain, read from the N-terminal end to the C-terminal end

• 2° structure: the ordered 3-dimensional arrangements (conformations) in localized regions of a polypeptide chain; refers only to interactions of the peptide backbone• e. g., -helix and -pleated sheet

• 3˚ structure: 3-D arrangement of all atoms• 4˚ structure: arrangement of monomer subunits with

respect to each other

1˚ Structure

• The 1˚ sequence of proteins determines its 3-D conformation

• Changes in just one amino acid in sequence can alter biological function, e.g. hemoglobin associated with sickle-cell anemia

• Determination of 1˚ sequence is routine biochemistry lab work (See Ch. 5).

2˚ Structure

• 2˚ of proteins is hydrogen-bonded arrangement of backbone of the protein

• Two bonds have free rotation:

1) Bond between -carbon and amino nitrogen in residue

2) Bond between the -carbon and carboxyl carbon of residue

• See Figure 4.1

-Helix

• Coil of the helix is clockwise or right-handed• There are 3.6 amino acids per turn• Repeat distance is 5.4Å• 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

-Helix (Cont’d)

-Helix (Cont’d)

• 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

-Pleated Sheet

• Polypeptide chains lie adjacent to one another; may be parallel or antiparallel

• R groups alternate, first above and then below plane• Each peptide bond is s-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

-Pleated Sheet (Cont’d)

• Glycine found in reverse turns

• Spatial (steric) reasons

• Polypeptide changes direction

• Proline also encountered in reverse turns. Why?

Structures of Reverse Turns

-Helices and -Sheets

• Supersecondary structuresSupersecondary structures:: the combination of - and -sections, as for example• unitunit:: two parallel strands of -sheet connected by

a stretch of -helix• unitunit:: two antiparallel -helices• -meander-meander:: an antiparallel sheet formed by a series of

tight reverse turns connecting stretches of a polypeptide chain

• Greek keyGreek key:: a repetitive supersecondary structure formed when an antiparallel sheet doubles back on itself

• -barrel-barrel:: created when -sheets are extensive enough to fold back on themselves

Schematic Diagrams of Supersecondary Structures

Fibrous Proteins

• Fibrous proteins:: contain polypeptide chains contain polypeptide chains organized approximately parallel along a single axis. organized approximately parallel along a single axis. TheyThey• consist of long fibers or large sheetsconsist of long fibers or large sheets• tend to be mechanically strongtend to be mechanically strong• are insoluble in water and dilute salt solutionsare insoluble in water and dilute salt solutions• play important structural roles in natureplay important structural roles in nature

• Examples areExamples are• keratin of hair and woolkeratin of hair and wool• collagen of connective tissue of animals including collagen of connective tissue of animals including

cartilage, bones, teeth, skin, and blood vesselscartilage, bones, teeth, skin, and blood vessels

Globular Proteins

• Globular proteins: proteins which are folded to a more or less spherical shape • they tend to be soluble in water and salt solutions• most of their polar side chains are on the outside and

interact with the aqueous environment by hydrogen bonding and ion-dipole interactions

• most of their nonpolar side chains are buried inside• nearly all have substantial sections of -helix and -

sheet

Comparison of Shapes of Fibrous and Globular Proteins

3˚ Structure

• The 3-dimensional arrangement of atoms in the molecule.

• In fibrous protein, backbone of protein does not fall back on itself, it is important aspect of 3˚ not specified by 2˚ structure.

• In globular protein, more information needed. 3k structure allows for the determination of the way helical and pleated-sheet sections fold back on each other.

• Interactions between side chains also plays a role.

Forces in 3˚ Structure

• Noncovalent interactions, including• hydrogen bonding between polar side chains, e.g., Ser

and Thr• hydrophobic interaction between nonpolar side chains,

e.g., Val and Ile• electrostatic attraction between side chains of opposite

charge, e.g., Lys and Glu• electrostatic repulsion between side chains of like

charge, e.g., Lys and Arg, Glu and Asp • Covalent interactions: Disulfide (-S-S-) bonds

between side chains of cysteines

Forces That Stabilize Protein Structure

3° and 4° Structure

• Tertiary (3°) structureTertiary (3°) structure:: the arrangement in space of all atoms in a polypeptide chain• it is not always possible to draw a clear distinction

between 2° and 3° structure

• Quaternary (4°) structureQuaternary (4°) structure:: the association of polypeptide chains into aggregations

• Proteins are divided into two large classes based on their three-dimensional structure• fibrous proteins• globular proteins

Determination of 3° Structure

• X-ray crystallography• uses a perfect crystal; that is, one in which all

individual protein molecules have the same 3D structure and orientation

• exposure to a beam of x-rays gives a series diffraction patterns

• information on molecular coordinates is extracted by a mathematical analysis called a Fourier series

• 2-D Nuclear magnetic resonance• can be done on protein samples in aqueous solution

High resolution method to determine 3˚ structure of proteins (from crystal)

Diffraction pattern produced by electrons scattering X-rays

Series of patterns taken at different angles gives structural information

Determines solution structure

Structural info. Gained from determining distances between nuclei that aid in structure determination

X-Ray and NMR Data

Myoglobin

• A single polypeptide chain of 153 amino acids• A single heme group in a hydrophobic pocket• 8 regions of -helix; no regions of -sheet• Most polar side chains are on the surface• Nonpolar side chains are folded to the interior• Two His side chains are in the interior, involved with

interaction with the heme group• Fe(II) of heme has 6 coordinates sites; 4 interact with

N atoms of heme, 1 with N of a His side chain, and 1 with either an O2 molecule or an N of the second His side chain

The Structure of Myoglobin

Oxygen Binding Site of Myoglobin

Denaturation

• Denaturation:Denaturation: the loss of the structural order (2°, 3°, 4°, 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• heat• large changes in pH, which alter charges on side

chains, e.g., -COO- to -COOH or -NH+ to -NH

• detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions

• urea or guanidine, which disrupt hydrogen bonding• mercaptoethanol, which reduces disulfide bonds

Denaturation of a Protein

Several ways to denature proteins• Heat

• pH

• Detergents

• Urea

• Guanadine hydrochloride

Denaturation and Refolding in Ribonuclease

Quaternary Structure

• Quaternary (4°) structureQuaternary (4°) structure:: the association of polypepetide monomers into multisubunit proteins• dimers• trimers• tetramers

• Noncovalent interactions• electrostatics, hydrogen bonds, hydrophobic

Oxygen Binding of Hemoglobin (Hb)

• A tetramer of two -chains (141 amino acids each) and two -chains (153 amino acids each); 22

• Each chain has 1 heme group; hemoglobin can bind up to 4 molecules of O2

• Binding of O2 exhibited by positive cooperativity; when one O2 is bound, it becomes easier for the next O2 to bind

• The function of hemoglobin is to transport oxygen• The structure of oxygenated Hb is different from that of

unoxygenated Hb• H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG) affect

the ability of Hb to bind and transport oxygen

Structure of Hemoglobin

Conformation Changes That Accompany Hb Function

• Structural changes occur during binding of small molecules

• Characteristic of allosteric behavior

• Hb exhibits different 4˚ structure in the bound and unbound oxygenated forms

• Other ligands are involved in cooperative effect of Hb can affect protein’s affinity for O2 by altering structure

Oxy- and Deoxyhemoglobin

Primary Structure Determination

How is 1˚ structure determined?

1) Determine which amino acids are present (amino acid analysis)

2) Determine the N- and C- termini of the sequence (a.a sequencing), and the Internal Residues

3) Determine the sequence of smaller peptide fragments (most proteins > 100 a.a)

4) Some type of cleavage into smaller units necessary

Primary Structure Determination

Protein Cleavage

Protein cleaved at specific sites by:

1) Enzymes- Trypsin, Chymotrypsin, Carboxypeptidases (C-terminus)

2) Chemical reagents

- Cyanogen bromide, cleaves at Methionine;

- PITC, cleaves from N-terminus (Edman Degradation)

- Hydrazine, cleaves from C-terminus

Enzymes which cleaves Internal Residues:

Trypsin- Cleaves @ C-terminal of (+) charged side chains (basic amino acid)

Chymotrypsin- Cleaves @ C-terminal of aromatics

Peptide Digestion

Cleavage by CnBr

Cleaves @ C-terminal of INTERNAL methionines

Determining Protein Sequence

After cleavage, mixture of peptide fragments produced.

• Can be separated by HPLC or other chromatographic techniques

• Use different cleavage reagents to help in 1˚ determination

Peptide Sequencing

• Can be accomplished by Edman Degradation

• Relatively short sequences (30-40 amino acids) can be determined quickly

• So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage

Peptide Sequencing