Transcript PPT File

高等生化學
Advanced Biochemistry
The Three-Dimensional
Structure of Proteins
陳威戎
Preface
Perhaps the more remarkable features of [myoglobin] are
its complexity and its lack of symmetry. The arrangement
seems to be almost totally lacking in the kind of
regularities which one instinctively anticipates, and it is
more complicated than has been predicted by any theory
of protein structure.
- J. Kendrew, article in Naure, 1958
Max Perutz,
1941-2002
John Kendrew, 1917-1997
Five themes to emphasize in this chapter
1. The three-dimensional structure of a protein is
determined by its amino acid sequence.
2. The function of a protein depends on its structure.
3. An isolated protein exists in one or few stable
structural forms.
4. The most important forces stabilizing the protein
structures are noncovalent interactions.
5. Common structural patterns that help us organize
our understanding of protein architecture.
Structure of the enzyme chymotrypsin, a globular protein
Internet resources for protein structures
Protein Data Bank, PDB (www.rcsb.org/pdb)
PDB ID: four-character identifier (ex: 6GCH)
Free molecular graphic programs:
RasMol, Chime, Swiss-Pdb Viewer
Protein Data Bank (PDB)
Research Collaboration for Structural Bioinformatics
http://www.rcsb.org/pdb/
Three-Dimensional Structure of Proteins
1. Overview of Protein Structure
2. Protein Secondary Structure
3. Protein Tertiary and Quaternary Structures
4. Protein Denaturation and Folding
Overview of Protein Structure
1. A protein’s conformation is stabilized largely
by weak interactions.
2. The peptide bond is rigid and planar.
1. A protein’s conformation is stabilized largely by
weak interactions.
Stability: the tendency to maintain a native conformation
Unfolded state: high degree of conformational entropy
Two simple rules:
(1) Hydrophobic residues are largely buried in the
protein interior, away from water.
(2) The number of hydrogen bonds within the protein is
maximized.
2. The peptide bond is rigid and planar
2. The peptide bond is rigid and planar
2. The peptide bond is rigid and planar
Ramachandran plot for L-Ala residues
Three-Dimensional Structure of Proteins
1. Overview of Protein Structure
2. Protein Secondary Structure
3. Protein Tertiary and Quaternary Structures
4. Protein Denaturation and Folding
Protein Secondary Structure
1. The a helix is a common protein secondary structure.
2. Amino acid sequence affects a helix stability.
3. The b conformation organizes polypeptide chains into
sheets.
4. b turns are common in proteins.
5. Common secondary structures have characteristic
bond angles and amino acid content.
1. The a helix is a common protein secondary structure
Knowing the right hand from the left
Five constraints affect the stability of an a helix
1. The electrostatic repulsion (or attraction) between
successive amino acid residues with charged R groups
2. The bulkiness of adjacent R groups
3. The interactions between R groups spaced three (or
four) residues apart
4. The occurrence of Pro and Gly residues
5. The interaction between amino acid residues at the
ends of the helical segment and the electric dipole
inherent to the a helix
2. Amino acid sequence affects a helix stability
Interactions between R
groups of amino acids
three residues apart in
an a helix
2. Amino acid sequence affects a helix stability
Helix dipole
3. The b conformation organize polypeptide
chains into sheets
3. The b conformation organize polypeptide
chains into sheets
4. b turns are common in proteins
4. b turns are common in proteins
5. Common secondary structures have characteristic
bond angles and amino acid content
5. Common secondary structures have characteristic
bond angles and amino acid content
5. Common secondary structures have characteristic
bond angles and amino acid content
Three-Dimensional Structure of Proteins
1. Overview of Protein Structure
2. Protein Secondary Structure
3. Protein Tertiary and Quaternary Structures
4. Protein Denaturation and Folding
Protein Tertiary and Quaternary Structures
1. Fibrous proteins are adapted for a structural function.
2. Structural diversity reflects functional diversity in
globular proteins.
3. Myoglobin provided early clues about the complexity
of globular protein structure.
4. Globular proteins have a variety of tertiary structures.
5. Analysis of many globular proteins reveals common
structural patterns.
6. Protein motifs are the basis for protein structural
classification.
7. Protein quaternary structures range from simple
dimers to large complexes.
8. There are limits to the size of proteins.
Fibrous proteins vs. globular proteins
Fibrous proteins
Globular proteins
Polypeptide
chain
Long strands or
sheets
Spherical or
globular shape
Secondary
structures
Single type
Several types
Protein
Functions
Support, shape
and external
protection
Enzymes and
regulatory
proteins
1. Fibrous proteins are adapted for a structural function
a-keratin
1. Evolved for strength.
2. Found in mammals, constitutes:
hair, wool, nails, claws, quills, horns, hooves,
and much of the outer layer of skin.
3. Part of intermediate filament (IF) proteins.
4. Right-handed a-helix.
5. Rich in hydrophobic residues: Ala, Val, Leu, Ile,
Met, and Phe.
6. Strength enhanced by covalent cross-links.
Structure of hair a-keratin
Cross section of a hair
Permanent waving is biochemical Engineering
Collagen
1. Evolved to provide strength.
2. Found in connective tissues such as:
tendons, cartilage, the organic matrix of bone,
and the cornea of the eye.
3. Left-handed triple helix, three a.a. per turn.
4. Three supertwisted polypeptides, a chains.
5. Typically contain: 35% Gly, 11% Ala, 21% Pro and
4-Hyp (4-hydroxyproline).
6. Repeating tripeptide unit: Gly-Pro-4-Hyp
Structure of collagen
Structure of collagen fibrils
Silk Fibroin
1. Fibroin, the protein of silk, is produced by insects
and spiders.
2. Predominantly in b conformation.
3. Soft and flexible.
4. Stabilized by extensive hydrogen bonding.
5. Rich in Ala and Gly.
Structure of silk
Strands of fibroin emerge from the spinnerets of a spider
Scurvy vs. Vitamin C (Ascorbic acid)
Why sailors, explorers, and college students should eat
their fresh fruits and vegetables!
Scurvy: caused by lack of vitamin C
small hemorrhages caused by fragile blood vessels,
tooth loss, poor wound healing, reopening of old
wounds, bone pain and degeneration, and
eventually heart failure.
Vitamin C: required for hydroxylation of proline and
lysine in collagen
Scurvy vs. Vitamin C (Ascorbic acid)
Vitamin C (L-ascorbic acid) is a white, odorless,
crystalline powder. It is freely soluble in water.
Recommended daily allowance: 60 mg (USA)
Scurvy vs. Vitamin C (Ascorbic acid)
Repeating tripeptide unit in collagen:
Gly-Pro-4-Hyp: Tm= 69℃
Gly-Pro-Pro: Tm= 41℃
Scurvy vs. Vitamin C (Ascorbic acid)
Prolyl 4-hydroxylase: a2b2 tetramer, each a sununit
contains one atom of nonheme iron (Fe2+)
2. Structural diversity reflects functional
diversity in globular proteins
3. Myoglobin provided early clues about the
complexity of globular protein structure
Tertiary structure
of sperm whale
myoglobin
The heme group
4. Globular proteins have a variety of tertiary structures
4. Globular proteins have a variety of tertiary structures
Methods for determining the three-dimensional
structure of a protein: X-ray diffraction
Methods for determining the three-dimensional
structure of a protein: X-ray diffraction
Protocols:
1. Protein over-expression and purification
2. Protein crystallization
3. X-ray diffraction
4. Phase determination and electron density maps
5. Model building and refinement
Advantages:
1. Best resolution
2. No size limitation (in contrast to NMR)
Limitations:
Technically very challenging to make crystals of proteins.
(heterogeneous samples, membrane proteins, protein complexes)
Methods for determining the three-dimensional structure
of a protein: Nuclear magetic resonance, NMR
Methods for determining the three-dimensional structure
of a protein: Nuclear magetic resonance, NMR
Protocols:
1. A concentrated aqueous protein sample (0.2-1 mM, 6-30 mg/mL)
labeled with 13C and/or 15N is placed in a large magnet.
2. An external magnetic field is applied; 13C and 15N nuclei will
undergo precession (spinning like a cone) with a frequency that
depends on the external environment
3. From these frequencies, computer determines the through-bond
(J coupling) and through-space (NOE) constants between every
pair of NMR-active nuclei.
4. These values provide a set of estimates of distances between
specific pairs of atoms, called "constraints“
5. Build a model for the structure that is consistent with the set of
constraints
Methods for determining the three-dimensional structure
of a protein: Nuclear magetic resonance, NMR
Advantages:
1. Native like conditions – sample is hydrated, not in a crystal lattice
2. Can get dynamic information – observe conformational changes
3. Can look at relative disorder of specific regions of a protein
– can see if a loop is static or flexible over time
Limitations:
1. Not as high resolution as x-ray
2. Require a lot of protein to get a good signal
3. Require very concentrated samples (can get insoluble aggregates)
4. Limit on protein size measurable, since molecule must tumble
rapidly to give sharp peaks. Typically, proteins must be <30kD.
5. Analysis of many globular proteins reveals
common structural patterns
1. The three-dimensional structure of a typical globular
protein can be considered an assemblage of polypeptide
segments in the a-helix and b-sheet conformations.
2. Supersecondary structures: motifs, folds
Stable arrangements of several elements of secondary
structure and the connections between them.
3. Polypeptides with more than a few hundred amino acid
residues often fold into two or more stable, globular units
called domains.
5. Analysis of many globular proteins reveals
common structural patterns
Structural domains in the polypeptide troponin C
Stable folding patterns in proteins
Burial of hydrophobic amino acid R groups so as to exclude water
requires at least two layers of secondary structures.
Stable folding patterns in proteins
Connections between elements of secondary structure cannot cross
or form knots.
Stable folding patterns in proteins
Two parallel b strands must be connected by a crossover strand.
Right-handed connections tend to be shorter and bend through
smaller angles, making them easier to form.
Stable folding patterns in proteins
Constructing large motifs from smaller ones
6. Protein motifs are the basis for protein
structural classification
SCOP databases in PDB.
6. Protein motifs are the basis for protein
structural classification
6. Protein motifs are the basis for protein
structural classification
6. Protein motifs are the basis for protein
structural classification
7. Protein quaternary structures range from
simple dimers to large complexes
Quaternary structure of deoxyhemoglobin
Rotational symmetry in proteins
Rotational symmetry in proteins
Rotational symmetry in proteins
Viral capsids
Three-Dimensional Structure of Proteins
1. Overview of Protein Structure
2. Protein Secondary Structure
3. Protein Tertiary and Quaternary Structures
4. Protein Denaturation and Folding
Protein Denaturation and Folding
1. Loss of protein structure results in loss of function.
2. Amino acid sequence determines tertiary structure.
3. Polypeptides fold rapidly by a stepwise process.
4. Some proteins undergo assisted folding.
1. Loss of protein function results in loss of function
Circular Dichrosim (CD) Spectroscopy - Introduction
1. When plane polarized light passes through a solution
containing an optically active substance the left and right
circularly polarized components of the plane polarized light
are absorbed by different amounts.
2. When these components are recombined they appear as
elliptically polarized light. The ellipticity is defined as q.
3. CD is the ellipticity (difference) in absorption between left and
right handed circularly polarized light that measured with
spectropolarimeter.
4. Proteins and nucleic acids contain elements of asymmetry
and thus exhibit distinct CD signals.
Far-UV (180-250 nm) CD for determining protein
secondary structure
Secondary Signal WL (nm)
Structure (+/-)
a-helix
b-sheet
random
coil
+
+
+
190-195
208
222
195-200
215-220
200
220
Near-UV (250-350 nm) CD is dominated by aromatic
amino acids and disulfide bonds
a.a. residue Abs max.
(nm)
254, 256
Phe
262, 267
Tyr
276
Trp
282
Disulfides
250-300
broad band
Circular Dichrosim (CD) Spectroscopy - Applications
1. Secondary structure content of macromolecules
2. Conformation of proteins and nucleic acids
- Effects of salt, pH, and organic solvents
3. Kinetics
- Protein folding, unfolding, denaturation or aggregation
4. Thermodynamics
- Protein stability to temperature or chemical denaturants
Circular Dichrosim (CD) Spectroscopy
2. Amino acid sequence determines tertiary structure
3. Polypeptide fold rapidly by a stepwise process
The thermodynamics of protein folding
depicted as a free-energy funnel
Death by misfolding: the prion diseases
1. A misfolded protein appears to be the causative agent of a
number of rare degenerative brain diseases in mammals.
2. Mad cow disease (bovine spongiform encephalopathy, BSE)
3. Related diseases:
Human- kuru, Creutzfeldt-Jakob disease (CJD)
Sheep- scrapie
Deer and Elk- chronic wasting disease
4. Typical symptoms: dementia and loss of coordination, fatal
Death by misfolding: the prion diseases
A stained section of the cerebral cortex from a patient with
Creutzfeld-Jakob disease shows spongiform degeneration.
Death by misfolding: the prion diseases
1. Prusiner S. provided evidence that the infectious agent has
been traced to a single protein (Mr 28,000), prion (PrP).
2. Role of PrP: molecular signaling function in brain tissues
3. Strains of mice lacking the gene for PrP suffer no ill effects.
4. Illness occurs when the normal cellular PrPc occurs in an
altered conformation called PrPSc.
4. Interaction of PrPSc with PrPc converts the latter to PrPSc,
initiating a domino effect in which more and more of the
normal cellular protein converts to the disease-causing form.
Death by misfolding: the prion diseases
The structure of human PrP in monomeric and dimeric forms.
4. Some proteins undergo assisted folding
Folding Accessory Proteins
4. Some proteins undergo assisted folding
Protein Disulfide Isomerase (PDI)
4. Some proteins undergo assisted folding
Peptidyl Prolyl Isomerase (PPI)
4. Some proteins undergo assisted folding
Molecular chaperones
4. Some proteins undergo assisted folding
Unrelated classes of chaperones
4. Some proteins undergo assisted folding- Chaperones
4. Some proteins undergo assisted folding- Chaperonins
Chaperonin: GroEL/GroES
4. Some proteins undergo assisted folding- Chaperonins
4. Some proteins undergo assisted folding- Chaperonins