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The Three-DimensionalStructure of Proteins
Part 1
Chapter 4
Proteins: Structure, Function, Folding
– 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
Learning goals: to Know:
Structure of Proteins
• Unlike most organic polymers, protein molecules adopt a specific three-dimensional 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 StructureThe 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 NMRTransverse 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
4th Edition: See pages 1075-1076, Fig 27-41
5th Edition: See pages 1107-1109, Fig 27-47, 48
Ubiquitin Targeting a Cytoplasmic Protein
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 sheet-like 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 KinaseExcludes Glycines – they are too flexible
Frequency of Amino Acids in 2o Structure
Circular Dichroism Spectra
A
Difference in right handed and left handed polarized light on the extinction coefficient.
• 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)
Protein Tertiary Structure
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 graphite-like 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.