3. Proteins 3D Structure Flashcards
Define
- protein conformation
arrangement in space of its constituent atoms which determine the overall shape of the molecule
Define
- native protein
Proteins in their natural state with intact structure that is not altered by heat, chemicals, enzyme reaction, or other denaturants are named “native proteins”.
Define:
- protein stability
the energy difference between the folded and unfolded state of the protein in solution. Remarkably, the free energy difference between these states is usually between 20 and 80 kJ/mol, which is of the magnitude of one to four hydrogen bonds.
Thus the stability of a protein is determined by large number of small positive and negative interaction energies.
Define:
- hydrophobic interaction
The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules
Define:
- hydrogen bond
a weak bond between two molecules resulting from an electrostatic attraction between a proton in one molecule and an electronegative atom in the other.
Define
- ionic interaction
- van der Waals interaction
- Ionic interaction
- type of linkage formed from the electrostatic attraction between oppositely charged ions in a chemical compound.
- Van der Waals interaction:
- Van der Waals forces are weak intermolecular forces that are dependent on the distance between atoms or molecules. These forces arise from the interactions between uncharged atoms/molecules.
- Weak attractions of two dipoles that bring the nuclei closer -Electron clouds will repel each other as nuclei are brought closer together
- At point where net attraction is maximal, nuclei are in van der Waals contact
- Each atom has a characteristic van der Waals radius (a measure of how close that atom will allow another to approach
- Describe how maximizing numerous weak interactions provides the dominant stabilizing force in protein structure.
- Crucial to macromolecular structure and function -
- Individually weak, but with a strong cumulative effect -
- For macromolecules, the most stable structure is usually that in which weak interactions are maximized -
- Folding of a polypeptide or polynucleotide chains into a 3D shape is determined by this principle -
- The binding of an antigen to a specific antibody depends on the cumulative effects of many weak interactions
The primary structure is held together by covalent peptide bonds. They are formed during the process of protein biosynthesis, where the amino acids lose one water molecule per reaction to attach to another amino acid.
The secondary structure is determined by hydrogen bonds between the main-chain peptide groups.
The tertiary structure is held by multiple types of bonds and forces, including hydrophobic interactions, hydrogen bonding, disulfide bridge, ionic bonding, as well as van der Waals forces. Among these forces, the non-specific hydrophobic interaction is the main force driving the folding of protein, while hydrogen bonds and disulfide bonds are responsible for maintaining the stable structure.
The quaternary structure is also stabilized by the non-covalent interactions and disulfide bonds as in the tertiary structure, where more than one polypeptide is held together to form a single functional unit called multimer.
- State two general rules about weak interactions underlying the structural patterns observed in soluble proteins.
1) Hydrophobic Interactions are critical hydrophobic residues are largely buried in the protein interior, away from water // polar aa usually on surface
2) Extensive H-bonding occurs within 2° structural units: the number of hydrogen bonds and ionic interactions within the protein is maximized, thus reducing the number of hydrogen-bonding and ionic groups that are not paired with a suitable partner -proteins within membranes and proteins that are intrinsically disordered or have intrinsically ordered segments follow different rules -this reflects their particular function or environment, but weak interactions are still critical structural elements
- Explain the chemical basis for the planarity of the peptide bond and the six atoms forming the peptide group.
- Peptide group has a rigid planar structure as a consequence of resonance interactions that give the peptide bond 40% double bond character
- The six atoms that are involved in the peptide bond share a common geometric plane since rotation around the carbon-nitrogen bond is restricted. This is due to the partial double bond character of the bond due to resonance
- Define the torsion angles φ and ψ in the polypeptide chain. Define the conventions for defining the angles of 0° and 180°
- Torsion angles (dihedral angles) or rotation angles:
- Calpha – N bond = φ (phi) F
- Calpha – C bond = ψ (psi) Y
By convention: both phi and psi are 180° when polypeptide chain is in fully extended conformation and increase clockwise viewed from Calpha
F and y defined as 0° when the two peptide bonds flanking that carbon are in the same plane (w/ atoms 1 and 4 in cis arrangement)
- Describe the physical limits on values of φ and ψ, and how those limits are illustrated in a Ramachandran plot.
- Sterically constrained
- Certain rotations around the psi or phi angles will cause amide hyrogen, carbonyl oxygen or substitutuents of Calpha to collide
- ie bring atoms closer than the van der waals distance
- Ramachandron plot:
- summarizes the sterically allowed values of phi and psi
phi/psi angles for aa in
- alpha helix = -57, -47
- Parallel Beta sheet: (f,y) near (-120°, +120°)
- Antiparallel Beta sheet (f,y) near (-140°, +140°) antiparallel
- Explain why proline and glycine have different Ramachandran plots from alanine.
Gly: the only residue without Cbeta atom = much less sterically hindered = permissible range of psi and phi covers larger area of ramachandran diagram
Pro: Cyclic side chain limits range of phi to around -60° = most conformationally restricted amino acid residue
- List the physical parameters of the α-helix, including characteristic φ and ψ angles, handedness and residues per turn.
- Right-handed
- ideally: φ (phi) = -57°
- ψ (psi) = -47°
- 3.6 residues per turn (5.4A/turn)
- R-groups point outward and downward from the helix
- Van der waals interactions and H-bonding stabilize
- H-bond between C=O of residue i and NH of residue i+4
Describe the main-chain hydrogen bonding pattern that stabilizes the α-helix.
Backbone hydrogen bonds are arranged such that the peptide C=O bond of the nth residue points along the helix axis toward the peptide N-H group of the (n+4)th residue
= STRONG H-BOND
- Describe the helix dipole and how it is generated by atoms in the polypeptide backbone.
A helix has an overall dipole moment due to the aggregate effect of the individual microdipoles from the carbonyl groups of the peptide bond pointing along the helix axis.
- Transmitted through intrachain H-bonds
Asp and Glu (-) are often found at the N-terminus (+) while Lys and Arg (+) are often found at the C-terminus (-) (charge complementarity).
- Phosphate groups (NAD/FAD) typically interact with Pos end (N-terminus)
- Outline different ways that the nature of amino acid R-groups can affect the stability of an α-helix.
The R groups of the amino acids stick outward from the α helix, where they are free to interact
- Ala, Glu and Met have highest propensity for alpha helix formation
- Asp and Glu (-) often at N-terminus (+)
- Lys and Arg (+) often at C-terminus (-) (charge complementarity)
- Pro and Gly have lowest
- Pro residues:
- introduce a kink
- proline is sometimes called a “helix breaker” because its unusual R group (which bonds to the amino group to form a ring) creates a bend in the chain and is not compatible with helix formation
Steric clashes between sequential large branched residues (Ile and Tyr) destabilize alpha helices
- Often flanked by Asn and Gln = sidechains fold back to form h-bonds == helix capping
The atoms in the helix backbone are packed densely in the core, stabilized by van der Waals interactions and hydrogen bonding.
Side chains project away from the helix core.
R-groups 3-4 residues apart in the primary
sequence may interact favourably in the helical form
Compare the structures of α-helices and β-sheets in terms of main-chain shape, hydrogen bonding pattern and location of R-groups.
B-sheet:
- H-bonding occurs between neighbouring polypeptide chains rather than within one (as in alpha helix)
- rippled or pleated (sheets formed by H-bonds between strands)
- Right handed
- R-groups extend perpendicularly to the plane of the sheet w/ successive chains on opposite sides
Alpha Helix
- Right-handed
- ideally: φ (phi) = -57°
- ψ (psi) = -47°
- 3.6 residues per turn
- R-groups point outward and downward from the helix
- H-bonding
- List the differences between parallel and antiparallel β-sheets.
- Antiparallel Beta Sheet:
- Neighboring hydrogen bonded polypeptide chains run in opposite directions
- (f,y) near (-140, +140)
- Parallel Beta sheet
- H-bonded polypeptide chains run in same direction
- (f,y) near (-120, +120)
Antiparallel ß sheets are slightly more stable than parallel ß sheets because the hydrogen bonding pattern is more optimal
Antiparallel sheets have more extended conformation
- Describe the shape and hydrogen bonding pattern of a β turn.
commonly observed features of beta turns are
- a hydrogen bond between the C=O of residue i and the N-H of residue i+3 (i.e, between the first and the fourth residue of the turn)
- Backbone H-Bond (characteristic)
- strong tendency to involve glycine and/or proline
- Always (almost) occur at protein surface
- Type I or Type II (differ by 180° flip of peptide unit) - characterized by combinations of f,y angles
- Irregular 2° structure.
- C=O of residue 1 to N-H of residue 4
- 180° change in direction of polypeptide.
- Can be used to connect antiparallel b-strands.
- Type II have glycine as the 3rd residue
Type I and Type II turns are the most common types of b-turns
Only Gly can adopt the necessary geometry (80°, 0°) at position i+2 (3rd position) for Type II turns
- State why Gly and Pro residues are frequently found in β-turns.
- Pro is often Residue 2 of both Type I and type II beta turns because it can assume the required conformation
- Proline is typically found in bends, unstructured regions between secondary structures.
- Gly:
- Type II turns: oxygen atom of residue two crowds the Cbeta atom = glycine (small)
- can adopt the necessary (80°, 0°) at i+2 (residue 3)
- Statistical analysis revealed that Pro and Gly residues are favored in β-turns presumably due to the cyclic structure of Pro and the flexibility of Gly
Define 3° structure, 4° structure, fibrous protein, globular protein.
Tertiary Structure
Tertiary Structure: Describes the folding of secondary structural elements
Quaternary Structure: the association of several protein chains or subunits into a closely packed arrangement.
Fibrous Protein: made up of elongated or fibrous polypeptide chains which form filamentous and sheet-like structures = structure
- repeating secondary structures
- eg keratin, collagen, elastin, and fibrin
Globular protein: a protein that is water-soluble and shaped like a sphere or a globe upon folding.
- may contain several types of 2° structure
- May have irregular structures (turns)
Define 3° structure, 4° structure, fibrous protein, globular protein.
- Define quaternary structure
Quaternary Structure: the association of several protein chains or subunits into a closely packed arrangement.
Tertiary Structure: Describes the folding of secondary structural elements
Fibrous Protein: made up of elongated or fibrous polypeptide chains which form filamentous and sheet-like structures = structure
- repeating secondary structures
- eg keratin, collagen, elastin, and fibrin
Globular protein: a protein that is water-soluble and shaped like a sphere or a globe upon folding.
- may contain several types of 2° structure
- May have irregular structures (turns)
Define 3° structure, 4° structure, fibrous protein, globular protein.
- Define fibrous protein
Fibrous Protein: made up of elongated or fibrous polypeptide chains which form filamentous and sheet-like structures = structure
- repeating secondary structures
- eg keratin, collagen, elastin, and fibrin
Tertiary Structure: Describes the folding of secondary structural elements
Quaternary Structure: the association of several protein chains or subunits into a closely packed arrangement.
Globular protein: a protein that is water-soluble and shaped like a sphere or a globe upon folding.
- may contain several types of 2° structure
- May have irregular structures (turns)
Define 3° structure, 4° structure, fibrous protein, globular protein.
- Define globular protein.
Globular protein: a protein that is water-soluble and shaped like a sphere or a globe upon folding.
- may contain several types of 2° structure
- May have irregular structures (turns)
Similarities (amongst soluble globular proteins):
- Mixtures of secondary structures (must include irregular structures along with regular secondary structures).
- Hydrophobic cores/hydrophilic exteriors.
- Closely-packed interiors.
- Maximized H-bonds in the interior.
Differences (between soluble globular proteins):
* Secondary structure composition.
* Prosthetic groups.
* Presence of disulfides (only in extracellular proteins).
Tertiary Structure: Describes the folding of secondary structural elements
Quaternary Structure: the association of several protein chains or subunits into a closely packed arrangement.
Fibrous Protein: made up of elongated or fibrous polypeptide chains which form filamentous and sheet-like structures = structure
- repeating secondary structures
- eg keratin, collagen, elastin, and fibrin
Compare and contrast α-keratins and collagen with respect to their secondary, tertiary and quaternary structures, typical amino acid content and type and nature of crosslinks formed.
Collagen
- strong, extensible, insoluble and chemically inert
- primary component of connective tissue (ECM protein - skin, bones, teeth)
- Three collagen strands form a parallel triple helix that is nearly one third glycine.
- The consensus amino acid sequence of collagen is (-Gly-Pro-Hyp-)n, where Hyp is 4-hydroxyproline.
- Collagen is heavily modified and cross-linked (at Lys and His), depending on the tissue type
Primary structure characterized by:
* glycine (allows chains to get close together)
- proline (stabilizes)
- 4-hydroxyproline (ensures appropriate conformation in collagen helix)
- also rich in 5-hydroxylysine (important for cross-linking
Composed of a regular, helical structure
* three residues per turn
* left-handed
* interchain H-bonds (h-bond between strands not within strands)
* three chains assemble into a right- handed super-helix
a-keratins
- enriched for alanine and cysteine (alpha helices and cross links)
- Structures are reinforced by disulfides – modifying the number of disulfides in different keratins makes the overall structures “harder” (more disulfides) or “softer” (fewer disulfides).
- The two chains in a dimer are twisted in a left-handed coiled-coil (superhelix)
- The helices of coiled coils are packed together by hydrophobic leucine ”zippers”, where leucine residues from one helix pack into the holes between side chains in the other helix.
- The interface between coiled coils is stabilized by ionic and polar interactions.
a-keratins
- enriched for amino acids ? and ? (alpha helices and cross links)
- Structures are reinforced by ?
- The two chains in a dimer are twisted in a ?
- The helices of coiled coils are packed together by ?
- The interface between coiled coils is stabilized by ? and ? interactions.
a-keratins
- enriched for alanine and cysteine (alpha helices and cross links)
- Structures are reinforced by disulfides – modifying the number of disulfides in different keratins makes the overall structures “harder” (more disulfides) or “softer” (fewer disulfides).
- The two chains in a dimer are twisted in a left-handed coiled-coil (superhelix)
- The helices of coiled coils are packed together by hydrophobic leucine ”zippers”, where leucine residues from one helix pack into the holes between side chains in the other helix.
- The interface between coiled coils is stabilized by ionic and polar interactions.
Collagen
- strong, extensible, insoluble and chemically inert
- primary component of ?
- Three collagen strands form a ? that is nearly one third ?.
- The consensus amino acid sequence of collagen is (-?-?-?-)n
- Collagen is heavily ? and ? (at Lys and His), depending on the tissue type
Primary structure characterized by:
* ? (allows chains to get close together)
- ? (stabilizes)
- ? (ensures appropriate conformation in collagen helix)
- also rich in ? (important for cross-linking
Composed of a regular, helical structure
* ? residues per turn
* ?-handed
* ? H-bonds (h-bond between strands not within strands)
* three chains assemble into a ?
Collagen
- strong, extensible, insoluble and chemically inert
- primary component of connective tissue (ECM protein - skin, bones, teeth)
- Three collagen strands form a parallel triple helix that is nearly one third glycine.
- The consensus amino acid sequence of collagen is (-Gly-Pro-Hyp-)n, where Hyp is 4-hydroxyproline.
- Collagen is heavily modified and cross-linked (at Lys and His), depending on the tissue type
Primary structure characterized by:
* glycine (allows chains to get close together)
- proline (stabilizes)
- 4-hydroxyproline (ensures appropriate conformation in collagen helix)
- also rich in 5-hydroxylysine (important for cross-linking
Composed of a regular, helical structure
* three residues per turn
* left-handed
* interchain H-bonds (h-bond between strands not within strands)
* three chains assemble into a right- handed super-helix
- Describe the structural information that can be determined using circular dichroism.
Circular dichroism (CD) spectroscopy is widely used to determine the amount of α-helix, β-pleated sheet, and random coil structures in a protein molecule. The principle of CD is based on the fact that asymmetrical structures absorb light in an asymmetrical manner
Circular dichroism:
* Depends on differential absorption of left and right circularly polarized light
* Sensitive to the secondaray structure of peptides and proteins
* A helices, b-sheets and random coil all have characteristic CD spectra
* Particularly usefull during denaturation/renaturation experiments
Define the terms supersecondary structure, motif, fold and domain.
Supersecondary Structure: a transitional bridge between the secondary and tertiary levels of protein structural organization
Motifs/Folds: Recognizable combinatiuons of secondary strucutre that appear in a number of different proteins
Domains: Discrete, independently folded compact units (may be composed of or include motifs) within a polypeptide // may have dif funtions within protein
Give one example of a simple motif that is repeated several times to create a common larger motif or domain.
The alpha/beta barrel is a common motif or fold constructed from repetitions of the beta-alpha-beta loop motif
Outline the connection between protein tertiary structure and evolutionary relationship within a protein family.
Proteins with similar tertiary structures are likely to have a common ancestor and thus share a common evolutionary history.
- similarity in tertiary structure is the result of the conservation of certain amino acid residues and their interactions over evolutionary time.
- Proteins may acquire mutations over time that alter their amino acid sequences and ultimately their tertiary structures
- conserved structural features may persist if they are critical to the protein’s function.
- eg enzymes within a protein family may have similar tertiary structures in their active sites, which are critical for substrate binding and catalysis.
Individual protomers in a complex may be related by ? or ? symmetry
Individual protomers in a complex may be related by rotational or translational symmetry
Rotational symmetry is the symmetry in which an object fits onto itself more than once while being rotated through. Translation symmetry is the symmetry in which an object moves from one position to another, with the same orientation in the forward and backward motion
Tetrahedral, octahedral, icosahedral symmetry
Tetrahedral, octahedral, icosahedral symmetry
- 12, 24, 60 protomers (respectively) arranged around multiple axes
What is Cyclic Symmetry?
Cyclic (C)
- Cn = N protomers arranged around 1 rotational axis
- Subunits are related by rotation about a single n-fold axis where n is the number of subunits
C2 = Twofold symmetry
C3 = Threefold symmetry
What is Dihedral Symmetry?
DN is 2N protomers arranged around 2 axes (2x CN)
- 1 axis is N-fold, the second is a twofold axis
- D2 = 4 protomers (2-fold x 2)
- D3 = 6 protomers (3-fold axis x 2-fold)
- D4 = 8 protomers (4-fold x 2)
Dihedral symmetry = subunits are related by rotation about a twofold axis which intersects with an n-fold axis at right angles
What is helical symmetry?
Helical Symmetry
- Protomers are related to each other by both rotation and translation
- Tobacco mosaic virus has helical symmetry
Describe how urea and guanidine hydrochloride denature proteins
In high concentrations
- Chaotropic agents (impact entropy)
- Water soluble -> interact with water -> ruin residue-water interactions
- Disrupt Hydrophobic interactions
- Solubolize hydrophobes
A chaotropic agent is a compound which disrupts hydrogen bonding in aqueous solution, leading to increased entropy
Describe how β-mercaptoethanol and dithiothreitol disrupt protein structure.
Reducing Agents
β-mercaptoethanol (BME) & Dithiothreitol (DTT)
- Reduce disulfide bonds
- Become oxidized as part of the reaction
The disulfide bonds, often present in secretory proteins and virtually absent in cytosolic proteins
Cytosol is reducing therefore these reducing agents will be unneccessary in cytosol/reducing environments
Describe Anfinsen’s experiment on denaturation and renaturation of ribonuclease, and relate this experiment to the tertiary structure being determined by amino acid sequence.
Anfinsen’s experiment on denaturation and renaturation of ribonuclease (RNase A) demonstrated that the three-dimensional tertiary structure of a protein is determined by its amino acid sequence.
- when ribonuclease is treated with a strong denaturant such as urea and beta-mercaptoethanol, it becomes unfolded, losing its three-dimensional structure and enzymatic activity. This unfolded state is known as the denatured state.
- when the denaturant was removed, the denatured ribonuclease spontaneously refolded into its native, active conformation, with all of its enzymatic activity restored. This process is known as renaturation.
- modifying the amino acid sequence of ribonuclease (mutations) = protein could no longer fold into its native conformation and lost its enzymatic activity, even in the absence of denaturants. This result suggested that the amino acid sequence alone determines the protein’s ability to fold into its native conformation.
Anfinsen’s experiment demonstrated that the tertiary structure of a protein is determined by its amino acid sequence, and that the** folding process is spontaneous and thermodynamically driven**. This principle is now known as the “anfinsen dogma.” It means that a protein’s native structure is the lowest free energy state among all possible conformations that can be adopted by the protein’s polypeptide chain.
Anfinsen’s experiment on denaturation and renaturation of ribonuclease provided evidence that the amino acid sequence of a protein determines its tertiary structure, and that the folding process is a spontaneous, energetically favorable process. This has profound implications for our understanding of protein structure and function, and has led to the development of numerous techniques for predicting and designing protein structures based solely on their amino acid sequences.
Describe a general model of protein folding.
Protein Folding:
- Cooperative process (just like unfolding)
- Not fully understood
Folding Pathways:
- Formation of 2° structures
- Formation of motifs
- Formation of domains
- Final 3° structure
After formation of 2° structure but prior to completion of 3° structure, protein adopts molten globule state
- Hydrophobic core (water pushed out)
- 2° structures present
- Collection of dynamic Structures
Describe a “molten globule”.
After formation of 2° structure but prior to completion of 3° structure, protein adopts molten globule state
- Hydrophobic core (water pushed out)
- 2° structures present
- Collection of dynamic Structures
Describe how the free energy and entropy of the polypeptide chain change during folding.
The folding process can be visualized as a free-energy funnel.
- Unfolded states (top) have large conformational entropy and relatively high free-energy.
- As folding progresses, the number of states present decreases and the conformational entropy decreases.
- Some structures may be relatively stable but do not represent the native structure
- The native, folded state (bottom) has the lowest conformational entropy.
Proteins fold into their correct minimal-energy configuartion because of the physicochemical properties of their amino acid sequence
KEY DIFFERENCE for misfolding
- Native state: High INTRAmolecular contact
- Misfolded proteins: HIGH INTERmolecular contact - stable structures
Describe how misfolding of proteins may be the cause of a disease.
KEY DIFFERENCE for misfolding
- Native state: High INTRAmolecular contact
- Misfolded proteins: HIGH INTERmolecular contact - stable structures ≠ Native structures
Accumulation of misfolded proteins can cause disease, and unfortunately some of these diseases, known as amyloid diseases (alzheimers, huntingtons), are very common
- Prion diseases
- Collagen defects
Intramolecular forces are those within the molecule that keep the molecule together, for example, the bonds between the atoms. Intermolecular forces are the attractions between molecules
Outline the general mechanisms of assisting protein folding by chaperones and the chaperonins.
Chaperones prevent aggregation and incorrect folding by binding to and stabilizing partially or totally unfolded protein polypeptides until the polypeptide chain is fully synthesized.
DnaK/DnaJ
- ATP binds to DnaK (Hsp70) which then associates with DnaJ (Hsp40) and misfolded substrate
- ATP hydrolysis changes the binding site for the protein substrate
- After release, GrpE is involved in nucleotide exchange (releases ADP)
Describe the reaction catalyzed by Protein Disulfide Isomerase (PDI).
PDI catalyzes the oxidation of dithiols and reduction of disulfide bonds and the isomerization of disulfide bonds
- catalyzes the shuffling of disulfide bonds to form the correct bonds of the native conformation
- Isomerase property - catalyze changes within one molecule
- Oxidoreductase property - enzymes that catalyze the transfer of electrons from one molecule (the oxidant, the hydrogen or the electron donor) to another molecule (the reductant, the hydrogen or electron acceptor)
Describe the reaction catalyzed by Peptide prolyl cis-trans isomerase (PPI).
Peptide Prolyl Isomerase (PPI)
- proline residues may be cis or trans peptide bonds (10% are cis)
- Adopting a cis bond is not spontaneous
- PPI assists in the reaction
- catalyzes the interconversion between the cis and trans conformations of proline residues in peptides and proteins
- involves the nucleophilic attack of a water molecule on the carbonyl carbon of the peptide bond, followed by the breaking of the bond between the proline residue and the preceding/following residue
- The proline residue then undergoes a rapid rotation around the peptide bond before reforming the bond with the preceding/following residue in the new conformation.
