Lecture 4 Flashcards
Big Picture Items
- Amino acids are the building blocks of proteins
- Stretches of proteins often fold into “α-helices” and “β-strands”
- α-helices and β-strands are building blocks of “domains”
- Domains are building blocks of proteins
- Fibrous and globular proteins differ in architecture
- Membrane proteins are often all-α helix or all-β strand
- Hydrophobic interactions play a key role in protein folding
- All proteins undergo continuous thermal motion
- Many proteins undergo functional conformational changes
Primary Structure
Linking amino acids by forming peptide units.
The order of the amino acids is called the “Primary Structure” of a protein
Secondary Structure
helix
tertiary structure
one complete protein chain (b chain of hemoglobin)
quarternary structure
the four separate chains of hemoglobin assembled into an oligomeric protein
Linking amino acids together via peptide bonds
An extended chain of a polypeptide
Emphasizing the planar peptide units (blue-greenish)
Two main chain torsional angles per residue:
phi (Φ) and psi (Ψ)
If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.
So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.
Each peptide unit contains six atoms: Cα, C, O, N, H and the next Cα.
The definition of phi (Φ)
If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.
So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.
The definition of psi (Ψ)
If one peptide unit is kept fixed, Φ and Ψ define the orientation of the second peptide unit.
So, in a first approximation, the course of a polypeptide chain is defined by a pair of dihedral angles (Φ and Ψ) per amino acid residue.
Secondary Structure Elements
• Proteins fold in complex ways with variations in Φ, Ψ angles per residue.
• In many instances, however, there are stretches of amino acids in a protein with a more regular structure
• These stretches have “secondary structure”
• In globular proteins, the major secondary structure elements
are:
• The α-helix
• The β-strand
The α-helix
VITAL CHARACTERISTICS: • 1.5 Å rise per residue • 3.6 residues per turn • 100 degrees RIGHT-HANDED rotation from residue to residue viewed along the axis • For all residues the same main chain dihedrals: Φ ≈ −570 and Ψ ≈ −470 • Hydrogen bonds between: • main chain C=O of residue n • the main chain NH of residue n+4
Right-handed and left-handed helices
A (discontinuous) helix is defined by:
- An object (e.g. a step from a staircase)
- The helix axis
- A rotation of the object about the helix axis
- A translation of the object parallel to the helix axis
Myoglobin
The predicted α-helix was confirmed when this protein structure was determined.
Myoglobin is an “all-α” soluble, globular protein. It contains eight α-helices.
Myoglobin also contains a “heme group” which contains an Fe(II)ion.
β-sheets: Antiparallel and parallel β-strands
NH to C=O hydrogen bonds between peptide units from different parts of the chain
Note the different pattern of hydrogen bonds in parallel versus antiparallel
β-sheets form a “pleated sheet”
In both parallel and anti-parallel β-sheets:
The side chains point alternatingly in opposite directions
There are also many MIXED ß-sheets, with some strands parallel and others antiparallel
β-sheets in globular proteins are often twisted
Carboxypeptidase A consists of a central MIXED β-sheet surrounded by α-helices.
The twist of the sheet is LEFT-handed when viewed in the plane of the sheet perpendicular to the β-strands.
This is due to the fact that when viewed along the β-strand, each strand has a RIGHT-handed twist.
This left-handed twist of the β-sheet is similar in parallel, anti-parallel and mixed β-sheets.
FIBROUS PROTEINS: Keratin and coiled coil α-helices
α-keratin is the principal protein of mammalian hair, nails, skin.
Keratin and coiled coil α-helices
The central 310-residue portion of α-keratin has a pseudo-repeat sequence a-b-c-d-e-f-g with nonpolar residues at a and d.
Note left-handed twist of theright-handed (!) α-helices.
In the coiled-coil structure the a:d’ and d:a’ contacts make a hydrophobic strip of contacts.
Fibrous Protein: The Collagen Triple Helix
Collagen: the most common vertebrate protein
A single collagen molecule consists of three
chains
Type I Collagen:
- Two α1 and one α2 chains
- Each chain about 1000 residues long
- Width: 14 Ǻ, length: ~ 3000 Ǻ
- About 30% of residues are Gly, 15-30% are 4 hydroxyPro
- Repeat of Gly-X-Y, often Gly-Pro-Hyp
- Three of these helices are wound around each other with a gentle, RIGHT-handed twist
- A Gly at every third position in the chain makes it possible to bring the chains very close together in the center of the triple helix.
The collagen triple helix
Interchain hydrogen-bonding pattern viewed along the collagen helix axis.
- Three consecutive residues from each chain shown in ball-and-stick
- Dashed lines are hydrogen bonds from Gly NH to Pro O in adjacent chains
- Dots show van der Waals surfaces.
- Note the close packing near the helix axis made possible by glycine residues at every third position.
Handedness summary
The α-helix:
The α-helix:
• RIGHT-handed, when viewed along the helix axis.
Handedness summary
In ß-sheets:
In ß-sheets:
• The individual ß-strand is slightly RIGHT-handed twisted, when viewed ALONG the length of the strand.
• In a ß-sheet, the strands are arranged LEFT-handed with respect to each other, when viewed IN the plane of the sheet PERPENDICULAR to the strands.
Handedness summary
In α-keratin:
In α-keratin:
• Each individual α-helix is RIGHT-handed
• The two helices are twisted around each other in a LEFT-handed manner.
Handedness summary
In collagen:
In collagen:
• The three strands are twisted around each other in a RIGHT-handed manner
Domains
Combinations of many secondary structure elements by turns and loops, and form a compact unit.
Triose Phosphate Isomerase (TIM)
A domain which occurs in a many proteins.
Note the “β-barrel” in the center surrounded by α-helices
Note the 8-fold repeated β-α motif
Membrane proteins are often either all-α or all-β
The protein avoids placing main chain C=O and NH groups in the hydrophobic bilayer)
Bacteriorhodopsin: α-HELICES crossing the membrane
OmpF Porin: β-BARREL crossing the membrane
Multi-domain proteins are very common
Combining domains is a way to to bring different functions of different domains together
N-terminal domain (gold) and C-terminal domain (blue) of the enzyme:
“Glyceraldehyde-3-phosphate dehydrogenase” (GAPDH)
(Actually only one subunit from the tetrameric protein GAPDH is shown)
C-terminal domain:
Substrate binding
N-terminal domain:
Cofactor binding
Proteins are only marginally stable:
A 100 residue protein is typically stable by only about 10 kcal (40 kJ) per mole.
Proteins have not evolved to be optimally stable, they have evolved to be optimally functional.
(Note: proteins are often modular, i.e. folded into multiple domains.
Therefore larger proteins are usually not more stable than smaller proteins)
Electrostatic effects:
Salt bridges between opposite charges relatively weak due to electrostatic screening by water
Disulfide bonds:
SS-bridges are an important stabilizing factor for extracellular proteins;
intracellular proteins very rarely have SS-bridges.
Hydrogen bonds:
Important to make H-bonds in native state since they are made with water in the unfolded state;
Native proteins almost never have unpaired donors/acceptors in the core!
Van der Waals
interactions:
Important to make these in the native state since they are made with water in the unfolded state
Conformational Entropy:
The protein has a much greater entropy in the unfolded than in the folded state!
Hydrophobic interactions:
Nonpolar sidechains come together in the folded protein to minimize the contact with water.
A major determinant of protein stability is the entropy gain of bulk water!
Protein denaturation (in vitro)
Loss of secondary, tertiary & quaternary structure without peptide bond hydrolysis.
Popular denaturants:
• Heat
• Guanidinium chloride (6M)
• Urea (8M)
Breaking SS bridges is not required for denaturation, but is required to arrive at a true random coil.
β-Mercaptoethanol (BME) is often used to reduce SS bridges.
Denaturation is often reversible!
Sequence determines the structure of a protein
The conclusion of Anfinson on protein denaturation-renaturation was that the primary sequence dictates the folded structure.
Hence: the secondary and tertiary structure is determined by the protein’s sequence.
Ribonuclease A
After denaturation, refolding needs careful attention regarding oxidation agents in order
to obtain correct disulphide bridges.
Myoglobin:
After denaturation and dialysis, it was found necessary to provide the heme moiety to
obtain native protein.
Muscle aldolase:
This tetrameric protein could be recovered in high yield after acid denaturation, showing
that assembly of the quaternary structure also follows from the protein’s sequence.
Folding pathways and energy landscapes in protein folding
Folding pathway
(hypothetical, yet
capturing current
thinking):
Proteins fold in a hierarchical manner. First, small local elements of secondary structure form.
Then, these coalesce
to yield larger
supersecondary
structure units.
These units coalesce with other units to form larger elements: domains and the complete folded chain.
Atoms are closely packed in the interior of a protein
Proteins are usually packed as tightly as organic crystals
However, there are two types of motion which are critical:
- Thermal motion around equilibrium positions of all protein atoms;
- Functional motions (“conformational change”) in response to
- encounters with other molecules
- changes in pH
Conformational Change: Calmodulin
Protein structure is important.
Yet, without functional conformational changes of proteins, life would be pretty miserable.
Atoms are Dynamic
Thermal motions around the equilibrium positions of atoms
Molecular Dynamics of Myoglobin
Calculated “snapshots” of myoglobin at intervals of 5 x 10-12 seconds superimposed
The protein is only represented by a Cα trace – blue lines
The heme group is yellow; a key histidine residue is gold.
Proteins are Dynamic
Thermal motions around the equilibrium positions of atoms can be surprisingly large as seen in a movie produced by a special purpose molecular dynamics computer called “Anton”.
