chapter 4 Flashcards
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 α-helices and β-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
Structure of the Peptide Bond
Structure of the protein is partially dictated by the properties of the peptide bond
The peptide bond is a resonance hybrid of two canonical structures
The resonance causes the peptide bonds
-to be less reactive compared to esters, for example
-to be quite rigid and nearly planar
-to exhibit a large dipole moment in the favored trans configuration
Resonance in the Peptide Bond
Each peptide bond has some double-bond character due to resonance and cannot rotate. Although the N atom in a peptide bond is often represented with a partial positive charge, careful consideration of bond orbitals and quantum mechanics indicates that the N has a net charge that is neutral or slightly negative.
The Rigid Peptide Plane and the Partially Free Rotations
-Rotation around the peptide bond is not permitted
-Rotation around bonds connected to the alpha carbon is permitted
-φ (phi): angle around the α-carbon—amide nitrogen bond
-ψ (psi): angle around the α-carbon—carbonyl carbon bond
-In a fully extended polypeptide, both ψ and φ are 180°
FIGURE 4-2b The planar peptide group. (b) Three bonds separate sequential α carbons in a polypeptide chain. The N—Cα and Cα—C bonds can rotate, described by dihedral angles designated Φ and Ψ, respectively. The peptide C—N bond is not free to rotate. Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge of the R groups.
Distribution of φ and ψ Dihedral Angles
-Some φ and ψ combinations are very unfavorable because of
-Some φ and ψ combinations are more favorable because of
-A Ramachandran plot shows the distribution of φ and ψ dihedral angles that are found in a
Some φ and ψ combinations are very unfavorable because of steric crowding of backbone atoms with other atoms in the backbone or side chains
Some φ and ψ combinations are more favorable because of chance to form favorable H-bonding interactions along the backbone
A Ramachandran plot shows the distribution of φ and ψ dihedral angles that are found in a protein
–shows the common secondary structure elements
–reveals regions with unusual backbone structure
Secondary Structures
-Secondary structure refers to a local spatial arrangement of the polypeptide backbone
-Two regular arrangements are common:
–The α helix
stabilized by hydrogen bonds between nearby residues
—The β sheet
stabilized by hydrogen bonds between adjacent segments that may not be nearby
-Irregular arrangement of the polypeptide chain is called the random coil
The α Helix
-Helical backbone is held together by
-_____-handed helix with
-Peptide bonds are aligned roughly
-Side chains
-Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n+4 amino acids
-Right-handed helix with 3.6 residues (5.4 Å) per turn
-Peptide bonds are aligned roughly parallel with the helical axis
-Side chains point out and are roughly perpendicular with the helical axis
The α Helix: Top View
-The inner diameter of the helix (no side chains) is about
-The outer diameter of the helix (with side chains) is
–Residues 1 and ___ align nicely on top of each other
-The inner diameter of the helix (no side chains) is about 4–5 Å
–Too small for anything to fit “inside”
-The outer diameter of the helix (with side chains) is 10–12 Å
–Happens to fit well into the major groove of dsDNA
-Residues 1 and 8 align nicely on top of each other
–What kind of sequence gives an α helix with one hydrophobic face?
Sequence affects
-Not all polypeptide sequences adopt
–Small hydrophobic residues such as __ and __ are strong helix formers
-Helix breakers?
-Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect
helix stability
-Not all polypeptide sequences adopt α-helical structures
-Small hydrophobic residues such as Ala and Leu are strong helix formers
-Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible, no N-H for H-bonding
-Gly acts as a helix breaker because the tiny R-group supports other conformations
-Attractive or repulsive interactions between side chains 3–4 amino acids apart will affect formation
-An α helix is not formed due to strong repulsion of adjacent
-Lys and Arg are positive at
Bulk and shape of ___,_____,____ all destabilize α helix if close together
–Two aromatic residues are often found
–The identity of residues near the end of the helix segment can affect the
-An α helix is not formed due to strong repulsion of adjacent Glu residues for a long block of Glu residues at pH 7
-Lys and Arg are positive at pH7 and do not form α helix
-Bulk and shape of Asn, Ser, Thr, Cys all destabilize α helix if close together
-Two aromatic residues are often found 3 residues away from each other resulting in a hydrophobic interaction
-The identity of residues near the end of the helix segment can affect the stability of an α helix.
The Helix Dipole
–Recall that the peptide bond has a strong
-All peptide bonds in the α helix have a
–The α helix has a large
-Negatively charged residues often occur near the
-Recall that the peptide bond has a strong dipole moment
–Carbonyl O negative
–Amide H positive
-All peptide bonds in the α helix have a similar orientation
-The α helix has a large macroscopic dipole moment
-Negatively charged residues often occur near the positive end of the helix dipole
Helix dipole. The electric dipole of a peptide bond is transmitted along
AA (-) often found near
Helix dipole. The electric dipole of a peptide bond is transmitted along an α-helical segment through the intrachain hydrogen bonds, resulting in an overall helix dipole. In this illustration, the amino and carbonyl constituents of each peptide bond are indicated by + and – symbols, respectively. Non-hydrogen-bonded amino and carbonyl constituents of the peptide bonds near each end of the α-helical region are shown in red.
AA (-) often found near N-terminus of the helical segment, AA (+) usually at C-terminus.
β Sheets (or β pleated Sheets)
-The planarity of the peptide bond and tetrahedral geometry of the α-carbon create a
-Sheet-like arrangement of backbone is held together by
-Side chains protrude from the sheet alternating in
-The planarity of the peptide bond and tetrahedral geometry of the α-carbon create a pleated sheet-like structure (zigzag)
-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
Parallel and Antiparallel β Sheets
-Parallel or antiparallel orientation of two chains within a sheet are possible
-In parallel β sheets the H-bonded strands run in the same direction
—Resulting in bent H-bonds (weaker)
-In antiparallel β sheets the H-bonded strands run in opposite directions
-Resulting in linear H-bonds (stronger)
β Turns
-β turns occur frequently whenever
-The 180°turn is accomplished over
–The turn is stabilized by a
-Proline in position
–β turns are often found near the surface of a
-β turns occur frequently whenever strands in β sheets change the direction
-The 180°turn is accomplished over four amino acids
-The turn is stabilized by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence
-Proline in position 2 or glycine in position 3 are common in β turns
-β turns are often found near the surface of a protein, where the central two AAs can H-bond with water
Most common types of beta turns
Type I and type II β turns are most common; type I turns occur more than twice as frequently as type II. Type II β turns usually have Gly as the third residue. Note the H-bond between the peptide groups of the first and fourth residues of the bends.
Proline Isomers
-Most peptide bonds not involving proline are in the trans configuration (>99.95%)
-For peptide bonds involving proline, about 6% are in the cis configuration. Most of this 6% involve β-turns
-Proline isomerization is catalyzed by proline isomerases
Circular Dichroism (CD) Analysis
-CD measures the
-Chromophores in the chiral environment produce
-CD signals from peptide bonds depend on
-CD measures the molar absorption difference Δε of left- and right-circularly polarized light: Δε = εL – εR
-Chromophores in the chiral environment produce characteristic signals
-CD signals from peptide bonds depend on the chain conformation
-can only determine secondary structure not tertiary structure
Protein Tertiary Structure
-Tertiary structure refers to the
-Stabilized by numerous
-Interacting amino acids are not necessarily
-Two major classes
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)
Fibrous Proteins
-Polypeptide chains arranged in
-Usually consist largely of a
-Functions:
-Polypeptide chains arranged in long strands or sheet
-Usually consist largely of a single type of secondary structure, the tertiary structure is relatively simple
-Functions: to provide support, shape, external protection to vertebrates
α keratin- an example of a ___ protein
–Part of the intermediate
–Fibrous proteins give
–Fundamental structure is a simple
-Insoluble due to
–Hydrophobic surfaces buried by
Fibrous protein
-Part of the intermediate filament protein family
Found in mammals in hair, wool, nails, horns, hooves and outer layer of skin
-Fibrous proteins give strength/flexibility to the structures in which they are found
-Fundamental structure is a simple repeating element of secondary structure
-Insoluble due to high concentration of hydrophobic residues both on the protein interior and surface
-Hydrophobic surfaces buried by packing many similar polypeptide chains to form elaborate supramolecular structures
structure of keratin in hair
single=right handed helix
two chain- coiled coil left handed sense
then it becomes protofilament
then it aggregates more to become protofibril
IGURE 4–11a Structure of hair. (a) Hair α-keratin is an elongated α helix with somewhat thicker elements near the amino and carboxyl termini. Pairs of these helices are interwound in a left-handed sense to form two-chain coiled coils. These then combine in higher-order structures
called protofilaments and protofibrils. About four protofibrils—32 strands of α-keratin in all—combine to form an intermediate filament. The individual two-chain coiled coils in the various substructures also seem to be interwound, but the handedness of the interwinding and other structural details are unknown.
Structure of Collagen
–Collagen is an important constituent of
–Each collagen chain is a long
–Three collagen chains intertwine into a
–The triple helix has higher
–Many triple-helices assemble into a
-Collagen is an important constituent of connective tissue: tendons, cartilage, bones, cornea of the eye
-Each collagen chain is a long Gly- and Pro-rich left-handed helix
-Three collagen chains intertwine into a right-handed superhelical triple helix
-The triple helix has higher tensile strength than a steel wire of equal cross section
-Many triple-helices assemble into a collagen fibril
4-Hydroxyproline in Collagen
–Forces the proline ring into a
–Offer more hydrogen bonds between the
–The post-translational processing is
-Forces the proline ring into a favorable pucker
-Offer more hydrogen bonds between the three strands of collagen
-The post-translational processing is catalyzed by prolyl hydroxylase and requires α-ketoglutarate, molecular oxygen, and ascorbate (vitamin C)
Prolyl-4-hydroxylase Hydroxylates Protein as
Procollagen
The Cγ-endo conformation of proline and the Cγ-exo conformation of 4-hydroxyproline.
Hydroxyproline is necessary to keep some prolines in the “exo” form to allow the collagen triple to form
Hydroxylysine Cross Links
Collagen Triple Helix Strands
Silk Fibroin
–Fibroin is the main protein in
-sturcture?
-Small side chains allow the
-Structure is stabilized by
-Fibroin is the main protein in silk from moths and spiders
-Antiparallel β sheet structure
-Small side chains (Ala and Gly) allow the close packing of sheets
-Structure is stabilized by
–hydrogen bonding within sheets
–London dispersion interactions between sheets
Spider Silk
-Used for webs, egg sacks, and wrapping the prey
-Extremely strong material
—stronger than steel
—can stretch a lot before breaking
A composite material
–crystalline parts (fibroin-rich)
–rubber-like stretchy parts
Water-Soluble Globular Proteins
Polypeptide chains folded into a spherical or global shape
Often consist of several types of secondary structure
Examples: enzymes and regulatory proteins
Globular Protein Structures are
The most abundantproteininhumanblood
Are Compact and Varied
The most abundantproteininhumanblood plasma. Transports hormones, fatty acids, and other compounds, buffers pH, and maintainsosmotic pressure, among other functions.
Similarities in globular proteins
-Compact folding
-Hydrophobic residues oriented away from water and toward the interior of the protein
-Hydrophilic residues on the surface
-Hydrogen bonds, ionic and disulfide bonds help to stabilize molecule
Motifs (folds)
Specific arrangement of several secondary structure elements
All alpha-helix
All beta-sheet
Both
B-a-B loop
B barrel
Motifs can be found as reoccurring structures in numerous proteins
Proteins are made of different motifs folded together
Quaternary Structure
-Quaternary structure is formed by the assembly of individual polypeptides into a larger functional cluster (multisubunit or multimer; oligomer (a few subunit)
-Overall structure may be asymmetric if nonidentical subunits
-Deoxyhemoglobin- subunits are arranged with one α and one β subunit- describe as a tetramer or as a dimer of α β protomers
Protein Structure Methods: X-Ray Crystallography
Steps needed
-Purify the protein
-Crystallize the protein
-Collect diffraction data
-Calculate electron density
-Fit residues into density
Pros
-No size limits
-Well-established
Cons
-Difficult for membrane proteins
-Cannot see hydrogens
Protein Structure Methods: Biomolecular NMR
Steps needed
-Purify the protein
-Dissolve the protein
-Collect NMR data
-Assign NMR signals
-Calculate the structure
Pros
-No need to crystallize the protein
-Can see many hydrogens
Cons
-Difficult for insoluble proteins
-Works best with small proteins
Protein Structure Methods: Cryo Electron Microscopy
prepare sample, freeze grid, collect images, image processing, reconstruction, structural analysis, model
Intrinsically Disordered Proteins
-Contain protein segments that
-Composed of amino acids whose
-Disordered regions can conform to
-Contain protein segments that lack definable structure
-Composed of amino acids whose higher concentration forces less-defined structure
Lys, Arg, Glu, and Pro
-Disordered regions can conform to many different proteins, facilitating interaction with numerous different partner proteins
Proteostasis
-Proteostasis: the continual maintenance of the active set of cellular proteins requires under a given set of conditions.
-Maintenance of cellular protein activity is accomplished by the coordination of many different pathways.
Protein Stability and Folding
-A protein’s function depends on its
-Loss of structural integrity with
-Proteins can be denatured by:
-A protein’s function depends on its 3D-structure
-Loss of structural integrity with accompanying loss of activity is called denaturation
-Proteins can be denatured by:
-heat or cold
-pH extremes
-organic solvents
-chaotropic agents: urea and guanidinium hydrochloride
Forces that denature
Heat- abrupt loss of structure suggests co-operative unfolding over a narrow temp range
Urea, detergents, guanidine hydrochloride, organic solvents like acetone and alcohol disrupt hydrophobic interactions
pH extremes alter net charge, electrostatic repulsion and H-bond breakage
Ribonuclease Refolding Experiment
–Ribonuclease is a
-Urea in the presence of
-When urea and
-The sequence alone determines the
–Quite “simple” experiment, but so
-Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds
-Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease
-When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed
-The sequence alone determines the native conformation
-Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize
Renaturation of unfolded, denatured ribonuclease. Urea denatures the ribonuclease, and mercaptoethanol (HOCH2CH2SH) reduces and thus cleaves the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishing the correct disulfide cross-links
Ribonuclease Refolding Experiment
–Ribonuclease is a
-Urea in the presence of
-When urea and
-The sequence alone determines the
–Quite “simple” experiment, but so
-Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds
-Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease
-When urea and 2-mercaptoethanol are removed, the protein spontaneously refolds, and the correct disulfide bonds are reformed
-The sequence alone determines the native conformation
-Quite “simple” experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize
Renaturation of unfolded, denatured ribonuclease. Urea denatures the ribonuclease, and mercaptoethanol (HOCH2CH2SH) reduces and thus cleaves the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishing the correct disulfide cross-links
Polypeptides fold rapidly by a Stepwise Process
-E. coli can make a
–Is not a completely
–Generally local
–This is followed by
-E. coli can make a 100-residue protein molecule in 5 seconds at 37ºC
-Is not a completely random process
-Generally local secondary structures form such as a helices
-This is followed by longer range interactions between the secondary structures to form stable super secondary structures
-Process continues till domains form and entire polypeptide is folded
Proteins folding follow a distinct path
A protein-folding pathway. A hierarchical pathway is shown, based on computer modeling. Small regions of secondary structure are assembled first and then gradually incorporated into larger structures. The program used for this model has been highly successful in predicting the 3D structure of small proteins from their amino acid sequence. The numbers indicate the amino acid residues in this 56 residue peptide that have acquired their final structure in each of the steps shown.
as folding progresses free energy
Large number of possible conformations so conformational energy is large
Folding progresses and the number of states decreases so there is decrease in free energy
Some Proteins Undergo Assisted folding
Not all proteins fold spontaneously as synthesized.
Chaperones: proteins facilitating correct folding pathways or providing microenvironments for folding.
Two major families: Hsp70 family and chaperonins.
Hsp70 (Heat shock proteins of Mr 70,000) are more abundant in cells stressed by elevated T.
Hsp70 function: assist protein folding, protect proteins from denaturation, or block the certain proteins that must remain unfolded; facilitate the quaternary assembly.
chaperones prevent
Chaperonins facilitate
Chaperones prevent misfolding
Chaperonins facilitate folding
Protein misfolding is the basis of
Protein misfolding is the basis of numerous human diseases
