This document discusses the structure of proteins at various levels: 1) Primary structure is the amino acid sequence of a polypeptide chain. 2) Secondary structure includes alpha helices and beta pleated sheets formed by hydrogen bonding between amino acids in the backbone. 3) Tertiary structure is the three-dimensional folding of the entire polypeptide chain, stabilized by interactions between amino acid side chains. 4) Quaternary structure refers to the association of multiple polypeptide subunits in a protein. The document outlines techniques like X-ray crystallography and NMR that are used to determine protein structures at high resolution.

1. Mary K. Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell
Chapter Four
The Three-Dimensional Structure of Proteins
Paul D. Adams • University of Arkansas

2. 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

3. 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., α-helix and β-pleated sheet
• 3˚ structure: 3-D arrangement of all atoms
• 4˚ structure: arrangement of monomer subunits with
respect to each other

4. 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).

5. 2˚ Structure
• 2˚ of proteins is hydrogen-bonded arrangement of
backbone of the protein
• Two bonds have free rotation:
1) Bond between α-carbon and amino nitrogen in
residue
2) Bond between the α-carbon and carboxyl carbon of
residue
• See Figure 4.1

6. α-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

7. α-Helix (Cont’d)

8. α-Helix (Cont’d)
• Several factors can disrupt an α-helix
• proline creates a bend because of (1) the restricted
rotation due to its cyclic structure and (2) its α-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

9. β-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

10. β-Pleated Sheet (Cont’d)

11. Structures of Reverse Turns
• Glycine found in reverse turns
• Spatial (steric) reasons
• Polypeptide changes direction
• Proline also encountered in reverse turns. Why?

12. α-Helices and β-Sheets
• Supersecondary structures: the combination of α-
and β-sections, as for example
• βαβ unit: two parallel strands of β-sheet connected by
a stretch of α-helix
• αα unit: two antiparallel α-helices
• β -meander: an antiparallel sheet formed by a series of
tight reverse turns connecting stretches of a
polypeptide chain
• Greek key: a repetitive supersecondary structure
formed when an antiparallel sheet doubles back on
itself
• β -barrel: created when β-sheets are extensive enough
to fold back on themselves

13. Schematic Diagrams of Supersecondary
Structures

16. Fibrous Proteins
• Fibrous proteins: contain polypeptide chains
organized approximately parallel along a single axis.
They
• consist of long fibers or large sheets
• tend to be mechanically strong
• are insoluble in water and dilute salt solutions
• play important structural roles in nature
• Examples are
• keratin of hair and wool
• collagen of connective tissue of animals including
cartilage, bones, teeth, skin, and blood vessels

17. 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 α-helix and β-
sheet

18. Comparison of Shapes of Fibrous and
Globular Proteins

21. 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.

22. 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

23. Forces That Stabilize Protein Structure

24. 3° and 4° Structure
• Tertiary (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°) 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

25. 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

26. X-Ray and NMR Data
High resolution method to determine 3˚
structure of proteins (from crystal)
Determines solution structure
Diffraction pattern produced by electrons
Structural info. Gained from
scattering X-rays
determining distances between
Series of patterns taken at different nuclei that aid in structure
angles gives structural information determination

27. Myoglobin
• A single polypeptide chain of 153 amino acids
• A single heme group in a hydrophobic pocket
• 8 regions of α-helix; no regions of β-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

28. The Structure of Myoglobin

29. Oxygen Binding Site of Myoglobin

30. 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 -NH3+ to -NH2
• detergents such as sodium dodecyl sulfate (SDS)
which disrupt hydrophobic interactions
• urea or guanidine, which disrupt hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds

31. Denaturation of a Protein

32. Denaturation and Refolding in
Ribonuclease
Several ways to denature
proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride

33. Quaternary Structure
• Quaternary (4°) structure: the association of
polypepetide monomers into multisubunit proteins
• dimers
• trimers
• tetramers
• Noncovalent interactions
• electrostatics, hydrogen bonds, hydrophobic

34. Oxygen Binding of Hemoglobin (Hb)
• A tetramer of two α-chains (141 amino acids each)
and two β-chains (153 amino acids each); α2β2
• 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

35. Structure of Hemoglobin

36. 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

37. Oxy- and Deoxyhemoglobin

38. 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

39. Primary Structure Determination

40. 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

41. Peptide Digestion

42. Cleavage by CnBr
Cleaves @ C-terminal of INTERNAL methionines

43. 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

44. 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

45. Peptide Sequencing