Conformational Properties of Polypeptide
Chains
Levels of Organization
• Primary structure
– Amino acid sequence of the protein
• Secondary structure
– H bonds in the peptide chain backbone
• α‐helix and β‐sheets
• Tertiary structure
– Non‐covalent interactions between the R groups within the protein
• Quanternary structure
– Interaction between 2 polypeptide chains
Why protein structure?
• In the factory of living cells, proteins are the workers, performing a variety of biological tasks.
• Each protein has a particular 3‐D structure that determines its function.
”Structure implies function”.
• Structure is more conserved than sequence.
• Protein structure is central for understanding protein functions. Sequence
Structure
Function
The basics of protein
• Proteins have one or more polypeptide chains
• Building blocks: 20 amino acids.
• Length range from 10 to 1000 residues.
• Proteins fold into 3‐D shape to perform biological functions.
Anfinsen’s thermodynamic hypothesis
“The three‐dimensional structure of a native protein
in its normal physiological milieu (solvent, pH, ionic
strength, presence of other components such as metal ions or prosthetic groups, temperature, etc.)
is the one in which the Gibbs free energy of the
whole system is lowest; that is, that the native conformation is determined by the totality of interatomic interactions and hence by the amino
acid sequence, in a given environment.”
--- Anfinsen’s Nobel lecture, 1972
Gibbs free energy
• A thermodynamic potential that measures the "useful"
or process-initiating work obtainable from an
isothermal, isobaric thermodynamic system.
• The maximum amount of non-expansion work that
can be extracted from a closed system
• Gibbs free energy (ΔG) or available energy is the
chemical potential that is minimized when a system
reaches equilibrium at constant pressure and
temperature.
Common structure of Amino Acid
Cα is the chiral
center
Amino
H
H
+
N
R
Side chain = H,CH3,…
Atoms numbered β,γ,δ,ε,ζ..
Cα
H
H
Backbone
O
C
-
O
Atom lost during
peptide bond
formation
Carboxylate
Amino Acids
Hydrophilic
Hydrophobic
The peptide bonds
Coplanar atoms
Primary structure
• The amino acid sequences of polypeptides chains.
Shape = Amino Acid Sequence
• Proteins are made of 20 amino acids linked by peptide bonds
• Polypeptide backbone is the repeating sequence of the N‐C‐C‐N‐C‐C… in the peptide bond
• The side chain or R group is not part of the backbone or the peptide bond
• The amino acid sequence (the primary structure) of a protein determines its three‐dimensional structure, which in turn determines its properties.
1˚ structure = sequence of amino acids
Secondary structure
• Local organization of protein backbone: – α‐helix
– β‐strand (which assemble into β‐sheet)
– turn and interconnecting loop.
Protein Folding
• The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions
• Weak non‐covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape
Protein Folding
• Proteins shape is determined by the sequence of the amino acids
• The final shape is called the conformation and has the lowest free energy possible
• Denaturation is the process of unfolding the protein
– Can be down with heat, pH or chemical compounds
– If the chemical compounds are removed, the proteins can be renatured or refold
Refolding
• Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner
What drives protein folding?
• Hydrophobic effects
– Hydrophobic residues tend to be buried inside
– Hydrophilic residues tend to be exposed to solvent
• Hydrogen bonds help to stabilize the structure.
Non‐covalent Bonds in Proteins
Globular Proteins
• The side chains will help determine the conformation in an aqueous solution
Hydrogen Bonds in Proteins
• H‐bonds form between 1) atoms involved in the peptide bond; 2) peptide bond atoms and R groups; 3) R groups
Protein Conformation
Framework
• Bond rotation determines protein
folding, 3D structure
Bond Rotation
Bond Rotation Determines Protein Folding
Protein Conformation
Framework
• Bond rotation determines protein
folding, 3D structure
• Torsion angle (dihedral angle) τ
– Measures orientation of four linked
atoms in a molecule: A, B, C, D
Protein Conformation
Framework
• Bond rotation determines protein
folding, 3D structure
• Torsion angle (dihedral angle) τ
– Measures orientation of four linked
atoms in a molecule: A, B, C, D
– τABCD defined as the angle between
the normal to the plane of atoms A-BC and normal to the plane of atoms BC-D
Ethane Rotation
A
A
D
B
D
B
C
C
Protein Conformation
Framework
• Bond rotation determines protein
folding, 3D structure
• Torsion angle (dihedral angle) τ
– Measures orientation of four linked
atoms in a molecule: A, B, C, D
– τABCD defined as the angle between
the normal to the plane of atoms A-BC and normal to the plane of atoms BC-D
– Three repeating torsion angles along
protein backbone: ω, φ, ψ
Backbone Torsion Angles
Backbone Torsion Angles φ
and ψ
• Dihedral angle φ : rotation about the bond
between N and Cα
• Dihedral angle ψ : rotation about the bond
between Cα and the carbonyl carbon
φ and ψ angles
Backbone Torsion Angles ω
• Dihedral angle ω :
rotation about the peptide
bond, namely Cα1-{C-N}Cα2
• ω angle tends to be
planar (0º - cis, or 180 º trans) due to
delocalization of carbonyl
π electrons and nitrogen
lone pair
Backbone Torsion Angles
• φ and ψ are flexible, therefore rotation occurs
here
• However, φ and ψ of a given amino acid
residue are limited due to steric hindrance
• Only 10% of the {φ, ψ} combinations are
generally observed for proteins
• First noticed by G.N. Ramachandran
Consequences of double bond character
in the peptide bond
trans peptide bond
cis peptide bond
Still another consequence: in the cis form, the R groups in adjacent residues
tend to clash. Hence almost all peptide bonds in proteins are in the trans
configuration.
Planarity of the peptide bond
Psi (ψ) – the
angle of
rotation about
the Cα-C
bond.
Phi (φ) – the
angle of
rotation about
the N-Cα
bond.
The planar bond angles and bond
lengths are fixed.
Side chain properties
• Carbon does not make hydrogen bonds with water easily – hydrophobic
• O and N are generally more likely than C to H‐
bond to water – hydrophilic
• General amino acids groups:
– Hydrophobic
– Charged (positive/basic & negative/acidic)
– Polar
37
Unlike the peptide bond, free rotation occurs about the other two backbone
bonds, but steric interactions within the polypeptide still severely limit
plausible conformations, so that only certain combinations of phi and psi
angles are “allowed”
and between carbonyls
or combinations of
an R group, carbonyl
or amide group.
between R groups
180
not allowed
Ψ 0
allowed
-180
-180
0φ
180
Steric Hindrance
• Interference to rotation caused by spatial arrangement of atoms within molecule
• Atoms cannot overlap
• Atom size defined by van der Waals radii
• Electron clouds repel each other
Steric Hindrance
Most combinations of ψ and φ for an amino acid are not allowed because of steric collisions between the side chains and main chain.
Theoretically if the torsion angles all are 180o we have a purely trans system ( N, Cα, C’) if the torsion angles are all 0 we have the purely cis‐ arrangement.
Which is preferred?
For the peptide bond (ω) [Ci’ – Ni+1]
The trans is preferred 1000:1 Certain side chain conformations are energetically favorable—
Ethane
Valine
Which valine stagger is energetically the best?
The 1st one Why?
G.N. Ramachandran
• Used computer models of small polypeptides to systematically vary φ and ψ with the objective of finding stable conformations • For each conformation, the structure was examined for close contacts between atoms
• Atoms were treated as hard spheres with dimensions corresponding to their van der Waals radii
• Therefore, φ and ψ angles which cause spheres to collide correspond to sterically disallowed conformations of the polypeptide backbone
Ramachandran diagram
Most angle combinations (75%) excluded by steric hindrance
Dark green most favored
Steric exclusion: powerful organizing principle
Limited conformations favor protein folding,
favorable entropy of too many conformations opposes folding
Ramachandran Plot
QuickTime?and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime?and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
G.N. Ramachandran, 1963
φ, ψ and Secondary Structure
Name
φ
ψ
Structure
alpha-L
57
47
left-handed alpha helix
3-10 Helix
-49
-26
right-handed.
π helix
-57
-80
right-handed.
Type II helices -79
150
left-handed helices formed by polyglycine
and polyproline.
Collagen
-51
153
right-handed coil formed of three left handed
helicies
• The classical version of the
Ramachandran plot for (a)
alanine, (b) glycine.
• The fully allowed regions are
shaded; the partially allowed
regions are enclosed by a solid
line.
• The connecting regions enclosed
by the dashed lines are
permissible with slight flexibility of
bond angles (a,b).
• (c) for all 19 non-glycines and (d)
for glycine.
• Most regions show a 45 degree
slope rather than being parallel to
any of the axes (c)
• For glycine (d), the beta sheet
region is split into two distinct
maxima and the two most
populated regions (red), which
were predicted to be only just
permissible as shown in (b).
• There are five areas in the glycine
plot; two with psi 0 and three with
psi 180.
Referenced from Hovmöller (2002)
Random Coil
Following translation we have a primary amino acid sequence and it’s random‐coil ~~~~~~~~~~~~ ‐‐ just flopping around
folds to a 3D structure
Uses ‐‐ van der waals interactions Hydrophobic interactions
Salt Bridges H‐bonds
Electrostatics forces
Motifs of Protein Structure
• Packing hydrophobic side chains into the interior of the molecule • Creating a HYDROPHOBIC core and a HYDROPHILIC surface.
• The problem with creating such a hydrophobic core from a protein chain is– to bring the side chains into the core the main chain must also come into the interior. • The main chain is highly polar and therefore hydrophilic, with one hydrogen bond donor NH and one acceptor C = O for each peptide unit. • In a hydrophobic environment these main chain polar groups must be neutralized by the formation of hydrogen bonds. • This problem is solved very elegantly by the formation of regular secondary structure within the interior.
Secondary Structure
• The two very important secondary structures of proteins are:
– α‐helix
– β‐pleated sheet
• Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain.
5P2‐49
Protein Folding
• 2 regular folding patterns have been identified –
formed between the bonds of the peptide backbone
• α‐helix – protein turns like a spiral – fibrous proteins (hair, nails, horns)
• β‐sheet – protein folds back on itself as in a ribbon –globular protein
α‐Helix
• 3.6 amino acids per turn of helix.
• The (p), distance per turn, is 0.54 nm, and the rise, distance between each atom is 0.15 (0.54/3.6).
• The N‐H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid four
residues earlier.
• This repeated i + 4 –> i hydrogen bonding is the most prominent characteristic of an α‐helix
Characteristics of α‐Helix
• Although structurally ordered, isolated α‐helices are usually marginally stable in aqueous solution. • A helix forms quickly (10‐5 to 10‐7 sec) but can unravel almost as quickly. • Formation is generally independent of length, though unraveling isn’t. • The most common along the outside with one face out into solution and one into the hydrophobic core.
• Alpha‐helix have a hydrophobic and hydrophilic side
• Side chains to change from hydrophobic to hydrophilic with a periodicity of 3‐4 residues. α‐helix
• An α‐helix can, in theory, be right or left handed depending on the screw direction of the chain.
• Left handed ones don’t exist!!! • Because of steric interactions between side chains and CO groups
• ALWAYS right handed!
Properties of the alpha helix
• φ ≈ ψ ≈ −60°
• Hydrogen bonds
between C=O of
residue n, and
NH of residue
n+4
• 3.6 residues/turn
• 1.5 Å/residue rise
• 100°/residue turn
54
Fig. 4-2a, p.84
Helices have
electric dipoles.
The dipole moment in the α‐helix
• All the hydrogen bonds in an α‐helix point in the same direction ‐ along the helical axis. • The dipole moments are also aligned along the helical axis. • A partial net positive charge at the amino end and a negative charge at C‐terminal end.
• The α‐helix has a macrodipole moment of ~n * 3.5 Debye units. n = # residues
Preferred amino acids in α‐helices
Different side chains have a weak but definite preference either for or against being in an α‐helix.
Good helix formers:
Ala, Glu, Leu, Met
Poor helix formers:
Ser
Gly ‐small, flexible‐doesn’t like to be in fixed position
Try ‐big and bulky
Pro ‐ can’t enter the i Æ i + 4 groups because can’t H‐
bond
Proline and α‐helix • Amino acid side chains project out from the α‐helix and don’t interfere with it—except for proline. • The last atom of the proline side chain is bonded to the main chain N atom which forms a ring. • This prevents the N from contributing to a hydrogen bond an adding steric hindrance to the α‐helix conformation. • Proline fits very well into the first (N‐term) of an α‐helix • Proline in long α‐helices distorts the local geometry causing a BEND. • Pro’s are often referred to as helix breakers. Helical wheel
• α‐helices are amphipathic
• Non‐polar side chains along one side of the helical cylinder and polar residues along the remainder of its surface. • Such helices often aggregate with each other or with other non‐polar surfaces.
• The view is down the helix with the hydrophobic core being in the middle. • The helix repeats itself after 5 turns or 18 residues so the 19‐21 residues are offset.
Hydrophilic side Hydrophobic side
Variations on the classical α‐helix
• In natural proteins a slightly different geometry is seen. • The CO groups tend to point out away from the helix axis and the H‐bonds are consequently not as straight • So, φ ~ ‐62o and ψ ~‐41o instead of the classical ‐57 to ‐
60o and ‐47 to ‐50o
• This geometry is more stable than the classical α‐helix because it permits each CO oxygen to H‐bond to the NH of the i + 4 residue.
• Occurs when the chain is more loosely or more tightly coiled
• With hydrogen bonds to residues i + 5 (π‐helix) or i + 3 (310 – helix), respectively, NOT i + 4
310‐helix and π‐helix • Has 3 residues per turn and contains 10 atoms between the hydrogen bond donor and acceptor.
• Rare and usually occur at the ends of regular helices or as a single turn helices. • Not energetically favorable for the most part
• The backbone atoms are packed too tight in the 310 or too loose in the π helix that there is a hole in the middle.
Contrast of helix end views between α (squarish) vs 3-10
(triangular)
β‐sheet (pleated sheet)
• Two types of β‐sheet, parallel (same N to C direction) and anti‐parallel (opposite N to C direction) between polypeptide chains.
• β‐Sheet contains 2 amino acid residues per turn, and cannot form H‐bond between amide H and carboxyl O.
• Hydrogen bonds can only formed between adjacent polypeptide chains.
• Anti‐parallel form is more stable than parallel form.
64
The beta strand (& sheet)
65
φ ≈ − 135°
ψ ≈ +135°
H bond
β pleated sheet
B Sheet: Lewis Structure
N-term
C-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
HC
H N
C O
CH
N H
O C
HC
H N
C O
C-term
N-term
Antiparallel sheet
N-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
C-term
N-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
C-term
Parallel sheet
Topologies of β‐sheets
Properties of beta sheets
• Core of many proteins is the β sheet
• Form rigid structures with the H‐bond
• Formed of stretches of 5‐10 residues in extended conformation
• Parallel/aniparallel,
Parallel/aniparallel
contiguous/non‐contiguous
Turns and Loops
Secondary structure elements are connected by regions of turns and loops
• Turns – short regions of non‐α, non‐
β conformation
• Loops – larger stretches with no secondary structure. Often disordered.
– “Random coil”
– Sequences vary much more than secondary structure regions
• Common AA found at turns are:
– glycine: small size allows a turn
– proline: geometry favors a turn
•
Proline
C
HN
COOH
H
P
Globular Proteins
• Usually bind substrates (O2) within a hydrophobic cleft in the structure.
• Myoglobin and hemoglobin are typical examples of globular proteins.
• Both are hemoproteins and each is involved in oxygen metabolism.
5P2‐72
Globular Protein Structure: Patterns of Folding • Infinite number of globular protein folding, but some principles of protein folding are found.
– Made up of a number of domains ‐ a compact locally folded region of tertiary structure, which perform different functions of proteins.
– Two major kinds of folding patterns, α‐helic and β‐sheet.
73
Fig. 6.16
Myoglobin: 2o and 3o Aspects
• Globular myoglobin has 153 AA arranged in eight α‐
helical regions labeled A‐H.
• The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue.
• His E7 and F8 are important for locating the heme group within the protein and for binding oxygen.
• A representation of myoglobin follows with the helical regions shown as ribbons.
5P2‐75
Myoglobin: 2o and 3o aspects
Some helical regions
Heme group with iron (orange)
at the center
5P2‐76
The Heme Group
-
CH2CH2COO
OOC CH2CH2
N of His
F8 binds
to
fifth site on
the iron.
H3C
N
N
-
CH3
Fe(II)
H2C
N
CH
His E7 acts
as a ”gate”
for oxygen.
N
Pyrrole ring
CH3
CH3
CH CH2
5P2‐77
Binding Site for Heme
• Lower His bonds covalently to iron(II)
• Oxygen coordinates to sixth site on iron and the upper His acts as a “gate”
for the oxygen.
5P2‐78
3D Structure of Cytochrome c
Cytochrome C 의 3차구조
Fig. 4-15, p.94
Fibrous Proteins
• Proteins with an elongated or filamentous form, often dominated by a single type of secondary structure over a large distance • Fibrous proteins have a high concentration of α‐helix or β‐sheet. • Examples include:
•
a‐keratin
•
collagen
•
silk fibroin
5P2‐80
Fibrous Proteins Structural Materials of Cells and Tissues
• More than 1/3 or more of the body protein in large vertebrates.
• Functions include:
– External protection such as skin, hair, feather and nail.
– Structure and support, shape and form, tendons, cartilage and bone.
• Native conformations of fibrous proteins are stable proteins after isolated and will not be denatured or unfolded.
81
Keratins
• Two classes of keratin, α‐ and β‐keratins.
• α‐ Keratins is the major fibrous proteins of hair and wool.
• Each α‐keratin molecule contains over 300 residues in length and all are α‐helical.
• Two α‐keratin helices bond together by their hydrophobic R‐side groups.
• The higher amounts of AAs are serine, glutamine, glutamic acid and cysteine.
84
Structure of α‐Keratin
• Adjacent polypeptide chains also crosslinked by disulfide bonds.
• Disulfide bond patterns between are what determine whether human hair is straight or curly.
Coiled‐Coil α‐Helical Dimer of α
Keratin
Amphipathic α helices:
Residues a, d, a’ and d’
hydrophobic, other residues
more hydrophilic
β-Keratin
• Contains β-sheet structure.
• Are found mostly in birds and reptiles for
feather and scales.
87
Fibroin
• They are all anti‐parallel pleated sheet structure.
• Comprise almost glycine, alanine and serine AA.
Gly‐ala‐gly‐ala‐gly‐serine‐gly‐ala‐ala‐ gly‐
• This alternation allows the β‐sheet to fit together.
• Strong and inextensible.
• No intrachain hydrogen bond, only interchain hydrogen bonds.
• No cystine cross‐linkage presence.
• The fibers are very flexible, e.g. silk.
88
Structure of Silk Fibroin
• Silk made by silkworms and spiders.
• Composed of microcrystalline array of antiparallel beta pleated sheets where each strand has alternating Gly and Ala or Ser residues.
Collagen
• Collagen is about one third of total protein in large animals, and is the major constituent of tendons and skin.
• Basic unit is tropocollagen which consists of three polypeptide chains in left‐handed helix tightly coiled into a three‐strand rope in right‐handed helix, arranged in head to tail.
• Tropocollagen have total about 3000 AA residues (each polypeptide has 1000 AA residues), having of molecule weight of 300,000.
• Every third residue can be only glycine because of the bulky effect. 91
Composition and structure of Collagen
• The tropocollagen subunit is a rod about 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands, each of which is a left‐handed helix. • Twisted together into a right‐handed coiled coil, a triple helix, a cooperative quaternary structure stabilized by numerous hydrogen bonds. • Tropocollagen subunits spontaneously self‐assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. • There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices
• Prolines and hydroxyprolines cause it to be very tightly twisted
and very rigid
Structure of Collagen Fibers
• Collagen is the
most abundant
vertebrate protein
and the major
stress-bearing
component of
connective tissue
(bone, teeth,
cartilage, tendon)
and fibrous matrix
of skin and blood
vessels.
• 3 intertwined lefthanded helices
• 3.3 residues/turn
• Repeating Gly-X-Y
(X often Pro, Y
often Pro or
hydroxyPro)
Tropocollagen
The 3 collagen molecules are crosslinked by :
* H‐bonds ‐ very tight and do not stretch
* Crosslinks between lysines, both interchain and intrachain, due to oxidation by enzyme LYSYL OXIDASE. Occurs only in collagen.
Carbohydrates on hydroxylysines
Tropocollagen molecules are head‐to‐tail and are staggered. More crosslinks occur with age. Collagen – Amino Acid Composition
1) Glycine (30%)
2) Proline
3) Modified amino acids – hydroxyproline
‐ hydroxylysine
4) Glycosylation
Primary sequence of collagen
‐Gly‐Pro‐Met‐Gly‐Pro‐Ser‐Gly‐Pro‐Arg‐
‐Gly‐Leu‐Hyp‐Gly‐Pro‐Hyp‐Gly‐Ala‐Hyp‐
‐Gly‐Pro‐Gln‐Gly‐Phe‐Gln‐Gly‐Pro‐Hyp‐
‐Gly‐Glu‐Hyp‐Gly‐Glu‐Hyp‐Gly‐Ala‐Ser
‐Gly‐Pro‐Met‐Gly‐Pro‐Arg‐Gly‐Pro‐Hyp‐
‐Gly‐Pro‐Hyp‐Gly‐Lys‐Asn‐Gly‐Asp‐Asp‐
Glycosylation
Sugars added to hydroxylysine or hydroxyproline
Addition of disaccharides –
Glu‐Glu
Gal‐Glu
Gal‐Gal
Glycosylation influences function
Fibrils – tendons – low carbohydrate
Sheets – Lens cap, cornea – High carbohydrate
Cross‐links between Tropocollagen
1) Between two lysine residues
1) Enzyme – lysyl oxidase
2) Aldol condensation: enolate + carbonyl
3) Link – Aldol cross‐link
2) Between two hydroxylysines and a lysine residue
1) Hydroxyprridinium cross‐link: increase heat stability
• Higher in older animals Importance of Hydroxylation
• The principal residue to be hydroxylated in in proteins is proline. Lysine may also be hydroxylated.
• Catalyzed by prolyl 4‐hydroxylase, prolyl 3‐hydroxylase, and lysyl 5‐hydroxylase. • The reactions require iron (as well as molecular oxygen and α‐
ketoglutarate) to carry out the oxidation
• Use vitamin C to return the iron to its oxidized state. • Deprivation of ascorbate leads to deficiencies in proline hydroxylation
• Leads to less stable collagen, which can manifest itself as the disease scurvy. Processes occuring within intracellular membrane of ER, golgi
Synthesis of Pro‐α‐chain (RER)
Hydroxylation of selected prolines and lysines
Glycosylation of selected hydroxylysines
Triple helix formation with 3 Pro‐α‐chains
Secretion (collagen molecule)
Formation of Collagen Fiber