Chapter 4
The Three-Dimensional
Structure of Proteins
Part 1
Proteins: Structure, Function, Folding
Learning goals: to Know:
–
–
–
–
–
Structure and properties of the peptide bond
Structural hierarchy in proteins
Structure and function of fibrous proteins
Structure analysis of globular proteins
Protein folding and denaturation
Structure of Proteins
• Unlike most organic polymers, protein
molecules adopt a specific threedimensional conformation.
• This structure is able to fulfill a specific
biological function
• This structure is called the native fold
• The native fold has a large number of
favorable interactions within the protein
• There is a cost in conformational entropy of
folding the protein into one specific native
fold
Favorable Interactions in Proteins
• Hydrophobic effect
– Release of water molecules from the structured solvation layer
around the molecule as protein folds increases the net entropy
• Hydrogen bonds
– Interaction of N-H and C=O of the peptide bond leads to local
regular structures such as -helices and -sheets
• London dispersion
– Medium-range weak attraction between all atoms contributes
significantly to the stability in the interior of the protein
• Electrostatic interactions
– Long-range strong interactions between permanently charged
groups
– Salt-bridges, esp. buried in the hydrophobic environment strongly
stabilize the protein
Folding  Final Structure: Chymotrypsin and
Glycine
75 Daltons
21,000 Daltons, 3 polypeptides
Protein Conformation
Stabilized by weak bonds:
ΔG separating folded and unfolded is small
Bond: H bonds, hydrophobic interatction, ionic
interaction and –S-S-.
Proteins possess a “solvation layer”
The extent of which depends on surface amino
acid R groups
Overall structural patterns:
Hydrophobic areas buried in protein interior
Number of H-bonds is maximized
The Peptide Bond
Only non-planar Bonds Rotate
Each α-Carbon has a Φ and Ψ
Getting the Angles
Many Angles are Prohibited due to Steric Overlap
Ramachandran Plot
Pauling and Corey and the Alpha Helix
How To Determine Protein Structure
The Classic Method – X-ray Crystallography
Methods to form Crystals take Proteins to their Solubility Minimum
Proteins Crystals in Electron Microscopy
X-ray Diffraction
X-ray Diffraction Pattern of Myoglobin and DNA
Fourier Transform to convert X-ray pattern to Electron Density Map
X ray Diffraction Patterns from Different Proteins
Fitting Electron Density Map to Structure
Fitting Electron Density Map to Primary Structure
X-ray Crystallography at Fine Resolution
Proton Nuclear Magnetic Resonance of a Protein
2 Dimensional NMR
NMR Structure of Myoglobin
NMR is limited to
small proteins
TROSY NMR
Transverse Relaxation Optimized Spectroscopy  increases
time overwhich NMR signals from neighboring methyl groups can
be detected. The trick is to deuterate the protein then protonate
methyls….
A look into a Proteasome cavity.
This protein is 670 kD! (20S)
Red groups = methyls that are
mobile
Yellow groups = active site…protein
degradation.
C+E News Feb 5, 2007
Here Is How It Worked
Nature 445:618 Feb 8, 2007
Proteasome Function
Core
Proteasome
Ubiquitin Binding Sites top and bottom
Ubiquitin Targeting a
Cytoplasmic Protein
4th Edition: See pages 10751076, Fig 27-41
5th Edition: See pages 11071109, Fig 27-47, 48
Protein AminoTerminal-aa
Half-life
stabilizing
M, G, A, S, T, V
>20 hrs
destabilizing
I, N, Y, D, P, L, F, K, R
30 – 2 min
What’s New: Free-Electron Lasers
X-ray pulses,
in series of
femtoseconds
on drops
containing
microcrystals.
Free-electron
X ray source
at Stanford
only one ($300
million),
Resolution 2Å.
Schichting, I. May, 2012 Max-Plank Gesellschaft
Alpha Helix
Stabilized by H-bonding to every 4th Amino Acid
Alpha Helix Stabilized by Dipole Moments and
Hydrogen Bonds Can be Right or Left Handed
Alpha Helix H-bonding - Stability
H-bonds to every 4th amino acid
So, amino acid-8 in a 15 aa α-helix is H-bonded to aa-11 and aa-5.
What about amino acids at the N- and C-terminal ends?
Instability is brought about by:
1. electrostatic repulsion or attraction – charged R groups.
2. adjacent bulky R groups.
3. interaction with R groups 3-4 aa’s up or down the helix.
4. occurrence of G.
5. interaction of aa’s at C-terminal and N-terminal ends with any
near aa-R group.
table 4-1
A
How Long is an 80 amino acid alpha helix?
FACTS to KNOW: One turn: 3.6 aa’s, 5.4 Å long
NOW DO IT
How Long is an 80 amino acid alpha helix?
FACTS to KNOW: One turn: 3.6 aa’s, 5.4 Å long
80 aa’s / (3.6 aa’s/turn) = 22.2 turns
22.2 turns x 5.4 Å/turn = 120 Å long
 Sheets
• The planarity of the peptide bond and tetrahedral
geometry of the -carbon create a pleated sheetlike structure
• Sheet-like arrangement of backbone is held
together by hydrogen bonds between the backbone
amides in different strands
• Side chains protrude from the sheet alternating in
up and down direction
Beta (Pleated Sheet) Structure
Antiparallel Beta-turns
Antiparallel Beta-turns with Proline
Normal = trans
In Beta-turns, Proline is cis
Ramachandran Plot showing 2o Structures
Ramachandran Plot of Pyruvate Kinase
Excludes Glycines – they are too flexible
Frequency of Amino Acids in 2o Structure
Circular Dichroism Spectra
Difference in
right handed
and left
handed
polarized
light on the
extinction
coefficient.
A
Protein Tertiary Structure
• Tertiary structure refers to the overall spatial
arrangement of atoms in a protein
• Stabilized by numerous weak interactions between
amino acid side chains.
 Largely hydrophobic and polar interactions
 Can be stabilized by disulfide bonds
• Interacting amino acids are not necessarily next to
each other in the primary sequence.
• Two major classes
– Fibrous and globular (water or lipid soluble)
Fibrous Proteins:
From Structure to Function
Alpha Keratin Structure – Almost All Alpha Helix
Biochemists at the Hairdressers
Why a Permanent is not a Temporary!
Chicken Feather α-keratin
Carbonizing (under O2 free environment) makes the fiber into a graphitelike material = light weight, high strength, low cost polymer)
Silk Fibroin is almost all Beta Structure
Silk
A
Spinnerets of a Spider – SEM, Artificially Colored
Things to Know and Do Before Class
1. The peptide bond and why it is planar.
2. Rotation around the alpha carbon, Ramachandran Plot.
3. Basic idea of X-ray crystallography and how it is used to get
3-D structure.
4. Alpha helix and B-structure.
5. Circular Dichroism spectra: what they demonstrate.
6. Fibrous proteins that are essentially all alpha helix or beta
structure.
7. EOC problems 1-4. We will do some in class and a case
study with music.