The Three-Dimensional Structure of Proteins Part 1 Chapter 4.
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Transcript of The Three-Dimensional Structure of Proteins Part 1 Chapter 4.
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The Three-DimensionalStructure of Proteins
Part 1
Chapter 4
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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:
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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
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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
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Folding Final Structure: Chymotrypsin and Glycine
75 Daltons
21,000 Daltons, 3 polypeptides
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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
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The Peptide Bond
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Only non-planar Bonds Rotate
Each α-Carbon has a Φ and Ψ
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Getting the Angles
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Many Angles are Prohibited due to Steric Overlap
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Ramachandran Plot
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Pauling and Corey and the Alpha Helix
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How To Determine Protein StructureThe Classic Method – X-ray Crystallography
Methods to form Crystals take Proteins to their Solubility Minimum
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Proteins Crystals in Electron Microscopy
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X-ray Diffraction
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X-ray Diffraction Pattern of Myoglobin and DNA
Fourier Transform to convert X-ray pattern to Electron Density Map
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X ray Diffraction Patterns from Different Proteins
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Fitting Electron Density Map to Structure
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Fitting Electron Density Map to Primary Structure
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X-ray Crystallography at Fine Resolution
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Proton Nuclear Magnetic Resonance of a Protein
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2 Dimensional NMR
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NMR Structure of Myoglobin
NMR is limited to small proteins
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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
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Here Is How It Worked
Nature 445:618 Feb 8, 2007
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Proteasome Function
Core Proteasome
Ubiquitin Binding Sites top and bottom
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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
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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
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Alpha Helix
Stabilized by H-bonding to every 4th Amino Acid
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Alpha Helix Stabilized by Dipole Moments and Hydrogen Bonds Can be Right or Left Handed
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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.
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table 4-1
A
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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
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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
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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
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Beta (Pleated Sheet) Structure
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Antiparallel Beta-turns
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Antiparallel Beta-turns with Proline
Normal = trans In Beta-turns, Proline is cis
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Ramachandran Plot showing 2o Structures
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Ramachandran Plot of Pyruvate KinaseExcludes Glycines – they are too flexible
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Frequency of Amino Acids in 2o Structure
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Circular Dichroism Spectra
A
Difference in right handed and left handed polarized light on the extinction coefficient.
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• 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
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Fibrous Proteins: From Structure to Function
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Alpha Keratin Structure – Almost All Alpha Helix
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Biochemists at the Hairdressers
Why a Permanent is not a Temporary!
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Chicken Feather α-keratin
Carbonizing (under O2 free environment) makes the fiber into a graphite-like material = light weight, high strength, low cost polymer)
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Silk Fibroin is almost all Beta Structure
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Silk
A
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Spinnerets of a Spider – SEM, Artificially Colored
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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.