Advanced Biochemistry 高等生化學 The Three-Dimensional Structure of Proteins 陳威戎

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 Preface Max Perutz, John Kendrew,

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. Five themes to emphasize in this chapter

Structure of the enzyme chymotrypsin, a globular protein

Protein Data Bank, PDB ( PDB ID: four-character identifier (ex: 6GCH) Free molecular graphic programs: RasMol, Chime, Swiss-Pdb Viewer Internet resources for protein structures

Protein Data Bank (PDB) Research Collaboration for Structural Bioinformatics

1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

1. A protein’s conformation is stabilized largely by weak interactions. 2. The peptide bond is rigid and planar. Overview of Protein Structure

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

Ramachandran plot for L-Ala residues

1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

1. The  helix is a common protein secondary structure. 2. Amino acid sequence affects  helix stability. 3. The  conformation organizes polypeptide chains into sheets. 4.  turns are common in proteins. 5. Common secondary structures have characteristic bond angles and amino acid content. Protein Secondary Structure

1. The  helix is a common protein secondary structure

Knowing the right hand from the left

Five constraints affect the stability of an  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  helix

2. Amino acid sequence affects  helix stability Interactions between R groups of amino acids three residues apart in an  helix

2. Amino acid sequence affects a helix stability Helix dipole

3. The  conformation organize polypeptide chains into sheets

4.  turns are common in proteins

5. Common secondary structures have characteristic bond angles and amino acid content

1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

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. Protein Tertiary and Quaternary Structures

Fibrous proteins vs. globular proteins Fibrous proteinsGlobular proteins Polypeptide chain Long strands or sheets Spherical or globular shape Secondary structures Single typeSeveral types Protein Functions Support, shape and external protection Enzymes and regulatory proteins

1. Fibrous proteins are adapted for a structural function

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  -helix. 5. Rich in hydrophobic residues: Ala, Val, Leu, Ile, Met, and Phe. 6. Strength enhanced by covalent cross-links.  -keratin

Structure of hair  -keratin

Cross section of a hair

Permanent waving is biochemical Engineering

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,  chains. 5. Typically contain: 35% Gly, 11% Ala, 21% Pro and 4-Hyp (4-hydroxyproline). 6. Repeating tripeptide unit: Gly-Pro-4-Hyp Collagen

Structure of collagen

Structure of collagen fibrils

1. Fibroin, the protein of silk, is produced by insects and spiders. 2. Predominantly in  conformation. 3. Soft and flexible. 4. Stabilized by extensive hydrogen bonding. 5. Rich in Ala and Gly. Silk Fibroin

Structure of silk

Strands of fibroin emerge from the spinnerets of a spider

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:  2  2 tetramer, each  sununit contains one atom of nonheme iron (Fe 2+ ) Scurvy vs. Vitamin C (Ascorbic acid)

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

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

Protocols: 1. A concentrated aqueous protein sample (0.2-1 mM, 6-30 mg/mL) labeled with 13 C and/or 15 N is placed in a large magnet. 2. An external magnetic field is applied; 13 C and 15 N 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

7. Protein quaternary structures range from simple dimers to large complexes Quaternary structure of deoxyhemoglobin

Rotational symmetry in proteins

Viral capsids

1.Overview of Protein Structure 2.Protein Secondary Structure 3.Protein Tertiary and Quaternary Structures 4.Protein Denaturation and Folding Three-Dimensional Structure of Proteins

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. Protein Denaturation and 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 . 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 ( nm) CD for determining protein secondary structure Secondary Structure Signal (+/-) WL (nm)  -helix  -sheet random-200 coil+220

Near-UV ( nm) CD is dominated by aromatic amino acids and disulfide bonds a.a. residueAbs max. (nm) Phe 254, , 267 Tyr 276 Trp282 Disulfides 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 (M r 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 PrP c occurs in an altered conformation called PrP Sc. 4. Interaction of PrP Sc with PrP c converts the latter to PrP Sc, 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