Lecture 4 – Introduction to

Protein Structure (1)

Introduction
Proteins are the functional forms of
polypeptides.
n represent all levels of the hierarchy of
macromolecular structure (1o to 4o)
n protein structure defined by:
w chemical properties of the polypeptide chain.
w the environment.
Several distinct classes of proteins:
1. globular proteins (water-soluble).
2. fibrous proteins (water-insoluble).
3. proteins that associate with membranes.
n These differ by tendencies in amino acid sequence
and composition, but…
w can all be described using the same basic principles.
Proteins Built from Amino
Acids
There are 20 common amino acids.
n all are -amino acids:
w amino and carboxylic acid groups
separated by a single, C carbon.
n all are L-amino acids(except Glycine).
n predominantly zwitterions, at pH 7.
n distinguished by chemical nature of R,
w the ‘side chain’.
Proteins also have other
components:
n D-amino acids.
w e.g., bacterial antibiotics, such as
gramicidin.
n Covalent modifications, following
synthesis.
w Disulfide bonds common in eukaryotic
Adoption of the L-form
Structurally Significant
Consider a natural protein, such as
Rubredoxin:
n protein in sulfur-metabolizing bacteria.
w Fe-S complex shown in green and
yellow.
w constructed of L-amino acids.
Will Rubredoxin made of D-form
amino acids have an inverted
structure?
n in 1993, L and D-forms of Rubredoxin
were synthesized.
w structures: X-ray crystallography.
w they are exact mirror images.
n L- and D- HIV protease also
synthesized.
Amino Acids
Distinguished by chemical nature of the side-
chain.
w size and shape.
w charge.
w hydrogen-bonding ability.
w ability to form disulfide-bridges, etc.
Amino acids can be broadly classified into 5
groups:
1. Aliphatic: R = hydrocarbon side-chain.
2. Nonpolar: R = other hydrophobic side-chain.
3. Aromatic: R = aromatic ring.
4. Polar: R = uncharged, polar group.
5. Charged: R carries a charge in solution, at pH 7.
n note: other classification schemes are also in use...
The Hydrophobic Effect
Most proteins are amphipathic:
n they include both hydrophobic and hydrophilic
residues…
Folding generally results in a partitioning of
residues:
n b/w Aq and non-Aq environments.
n hydrophobic residues –
w will each ‘desire’ to avoid water:
w tend to reside in a membrane or protein interior.
n hydrophilic residues –
w will each ‘desire’ to interact with water:
w tend to remain hydrated, reside on a protein exterior.
This partitioning b/w Aq and non-Aq
environments:
n leads the hydrophobic effect, which drives protein
folding.
The Partition Coefficient
The Partition Coefficient, P:
n measures the partitioning of a residue between Aq and
non-Aq environments.
n Consider a two-solvent system…
w with separate Aq and non-Aq environments;
w that are in contact, at equal conditions (e.g.,
Temperature).
n P answers the question,
w ‘how much of a given amino acid will reside in each
environment?’
For a given amino acid, P is measured by:
P = nonaq /aq, where

w i = mole fraction residing in environment i.

n conceptually simple, but there are some practical

Hydrophobicity Scale
Numerous scales of amino acid hydrophobicity
have been proposed.
n most based on the Go to transfer from water to
octanol.
n which is ‘best’ is controversial, but one popular scale
is…
The Hydrophobicity of Fouchere and Pliska
(1983):
n hydrophobicity parameters,  derived from:
w transfer from water to octanol.
w N-acetyl-amino acid amides.
n Hydrophobicity parameter for a given amino acid:

 = ln P = ln (nonaq / aq)

w Then: Hydrophobic:  > 0; Hydrophilic:  < 0

Aliphatic Amino Acids
These amino acids have alkyl side-chains:
n so that R is a hydrocarbon.
All are hydrophobic (P > 1).
n hence, have hydrophobicity, = ln P > 0.
Hydrophobicity increases with side-chain
length:
n = 0.31, 1.22, 1.70, 1.80 for Ala, Val, Leu, and Ile,
respectively.
Nonpolar Amino Acids
Have a nonpolar side chain, other than a
hydrocarbon.
n (Almost) all are hydrophobic (> 0).
n no simple correlation with chain ‘length’.
w = 0, 0.72, 1.54, 1.23 for G, P, C, and M, respectively.
Otherwise, have more distinct characters:
n Glycine is achiral; very flexible (a ‘helix-breaker’).
n The side chain of Proline is a closed ring;
w strong influence on the nature of the peptide bond.
n In some proteins, Cysteine residues form disulfide links.
Aromatic Amino Acids
All highly hydrophobic.
n Phe has a hydrophobicity of  = 1.79.
n Tyr is less hydrophobic, at  = 0.96,
w due to its reactive hydroxyl group.
n Trp is the most hydrophobic residue ( = 2.25).
Rings bulky… and tend to interact with other
rings.
n due to pi-pi interaction.
n in Aq. solution, rings
perpindicular.
w entropically favored.
n This is in contrast with
the stacked rings in DNA…
w minimizes solvent
exposure.
Polar Amino Acids
Each contains groups with partial charges,
n and therefore, tend to form hydrogen bonds...
Thus, much less averse to water:
n Asn, Gln, Ser all have negative hydrophobicity values.
w  = -0.60, -0.22, and –0.04, respectively.
n Thr is slightly hydrophobic, at  = 0.26.
w in some scales, Thr is hydrophilic (e.g., in the ‘hydropathy’
scale).
Charged Amino Acids
Amino acids that carry a charge very hydrophilic:
n Lys and Arg (+) charged at pH 7
w very hydrophilic ( = -0.99 and  = –1.01, respectively).
n His can also be (+) charged at pH 7 (environment-
sensitive).
w intermediate hydrophobicity ( = 0.13).
n Aspartic acid and Glutamic acid (-) charged at pH 7
w very hydrophilic ( = -0.77 and –0.64, respectively).
Overall Charge of a Protein

In passing, we note that the overall protein

charge:
n depends on the number of acidic and basic
residues...
n at the experimental pH of interest.
The Isoelectric Point (pI)
n = the pH at which the total charge of a protein is
zero.
n Protein charge density can be estimated from pI:

c = (pI-pH)/MW

w here, MW is the molecular weight of the protein.

n For pH > pI, overall charge negative (deprotonation).
Protein Structure
Proteins may have up to 4 levels of structure:
n Primary structure:
w the N to C amino acid sequence of the polypeptide.
n Secondary structure:
w helices resulting from local folding.
n Tertiary structure:
w global folding of secondary structures into a larger
structure.
n Quaternary structure:
w association of several, independent polypeptides.
We will look at each, in detail…
n 1o and 2o Structure (this Lecture)
n 3o and 4o structure (next Lecture)
Protein Primary Structure
A polypeptide - covalently linked chain of amino
acids.
n each linked amino acid called a ‘residue’.
n each pair of residues connected by a peptide bond:

The N-terminal to C-terminal sequence of

residues:
n Is the primary structure (1o) of the encoded protein.
Anfinsen’s Principle

Anfinsen’s Principle is basic to biochemistry:

n ‘The information needed to fold a macromolecule into
its native, 3-D structure is contained in its sequence’.
w Denatured ribonuclease spontaneously refolds into the
enzymatically active form, in vitro(Anfinsen, 1963).
1o structure then specifies the higher structure:
n Each sequence corresponds to a well-defined 3-D
structure.
w or to a family of closely related structures with activity.
w the ‘native state’.
n On the other hand… a unique structure does not
require a unique sequence.
w level of sequence homology required for similar
structure is only about 25%-30%.
w non-homologous sequences can also have similar
structures.
 o
Protein 2 Structure: The
Helices of Polypeptides
Protein secondary structure (2o) –
n refers to the regular and repeating structure of a
polypeptide.
w here, regular means symmetric.
n A linear chain of chiral building blocks can form only one
symmetric structure…a helix.
w Note a -strand is also a helix.
Protein 2ostructure thus refers to the helices
formed by polypeptide chains.
n all are held together by hydrogen bonds.
w H-bond donors = amino-Hydrogens.
w H-bond acceptors = carboxyl-Oxygens.
n The importance of H-bonding to biopolymer stability:
w first recognized by Linus Pauling.
Traditional Names for Helices
Determined by the nature of the repeating H-
bond:
1. N = number of residues in 1 helix turn.
2. d = number of atoms in the ring formed by each H-
bond donor (amino-H) and acceptor (keto-O).
w each helix assigned the name, ‘Nd’.
w e.g., in a 310 helix,
n Each turn contains N=3 residues;
n each H-bonded donor/acceptor pair form a ring of d =
10 atoms.
This notation has several shortcomings:
w a -sheet cannot be described in these terms:
n each strand is a 2-fold helix, but H-bonds between
strands.
w No information about handedness (left or right).
Helical Symmetry
Helical symmetry refers to discrete screw
symmetry.
n residues rotate andrise in a repeating
manner along an axis.
n This is a special case of screw symmetry:
w symmetry refers to monomer positions,
at discrete values of .
For n-fold helical symmetry, monomer
positions related by:
Cn(x,y,z)i + T = (x,y,z)i+1

n Cn = n-fold rotational symmetry operator.

w T = resulting translation down the helix axis.
n (x,y,z)i= position of stair, i.
n total translation after n applications of C :
The Standard Terminology

For displacements down the helical axis:

w total rise/turn = pitch, P.
w rise/step = helical rise, h.
w steps/turn = helical repeat, c (= n).
P=ch
For angular displacements about the axis:
w angle/step =  = helical angle, or twist.
 = 2/c = 2h/P
For helical symmetry about the z-axis,
n Positions of adjacent steps then related by:
Helix Handedness
The Symmetry Matrix does not uniquely specify a
helix:
n a helix can be left or right-handed.
n in principle, handedness given by :
w right-handed:  > 0.
w left-handed: .
n but we have the convention:  >= 0.
w all rotations defined as right-handed.
w Our notation is degenerate…
We distinguish right and left helices by:
n the axial displacement: P or h.
w For right-handed helices, P and h > 0.
w For left handed helices, P and h < 0.
The helix shown here is right-handed.
Naming Helices by Symmetry
Helical symmetry is denoted by NT:
n N denotes its N-fold rotation operator.
n T = translation generated by the symmetry
operator, in repeats (monomers).
n compare with Nd notation.
Example: Take the helix at right...
n Each residue rotated by +120o.
w 3-fold helical symmetry (N = 3).
n 1 application of the symmetry operator:
w translates a residue by +1 repeat, or P/3.
w thus, T = 1.
n This helix has 31 symmetry.
w note that it is a right-handed helix.
w right-handed helices with integer N are N1 helices.
The 310 Helix has 31
Symmetry
Example: The 310 Helix
n one of the two common helices in
proteins.
n Commonly named by Nd notation:
w N=3 residues/turn.
w d=10 atoms in each H-bond closed
ring.
n note: green line includes a shared

H.
w ith keto-O H-bonded with (i+3)th
Nitrogen.
The 310 helix has 31 helical
symmetry.
n Each residue rotated by +120o.
w exactly 3 residues/turn.
The -helix has 185
Symmetry
The most common helix in globular
proteins.
n ith keto-O H-bonded with the (i+4)th Nitrogen.
w 13 atoms b/w H-bond donor and acceptor.

The -helix has 3.61 helical symmetry.

n Each residue rotated by +100o.
w 3.6 residues/turn (N = 3.6).
n 1 application of the symmetry operator:
w translates a residue by +1 repeat, or P/3.6.
w thus, a right-handed helix, with  .

For helices with non-integral symmetry:

n N and T converted to integers…
n -helix said to have 185 symmetry.
w 18 residues in 5 full turns.
Left-Handed Helices
How do we construct a left-handed helix…
n using only right-handed rotations?
Consider the helix at right:
n Each residue rotated by +120o (N=3).
n But, application of this operator:
w translates each residue by 2 repeats (T = 2).
n The resulting helix (units 1,2,3):
w has 32symmetry.
w has gaps at 1’, 2’, 3’.
Copying this unit a distance P along the
axis:
n fills the gaps, generating a left-handed
helix…
…but, using only right-handed rotations.
A left-handed helix with N-fold helical
symmetry:
The ‘trans-conformation’ is a 21
helix
Consider the fully extended
polypeptide:
n keto-O and amino-H of each Peptide bond in
the trans configuration, and…

n For each residue, both and  = 180o.

w …remember our convention:
n Polymer chemistry: cis = 0o.
w Biopolymer in a ‘fully’ trans conformation…
n Somewhat expanded use of the term, ‘trans’.
n …since cis/trans defined for configurations.
The -strand is also a 21 Helix

Differs from the trans-

conformation:
n a -strand is pleated, like a curtain.
n shorter (smaller rise, h).
In order to be stable, -strands
combine to form sheets:
n by strand-to-strand hydrogen
bonding.
n two general types:
w anti-parallel sheets (A).
w parallel sheets (P).

Generally, these sheets are

twisted.
Standard Helices of
Biopolymers

n these include all of the standard, 2o structures of

biopolymers.
Thus, our original contention is correct:
n the only symmetric building block formed by a chain
of chiral units is a helix.
Question: can the backbone adopt these
structures?
The Peptide Bond

The peptide bond…

n links each pair of adjacent residues;
n is an amide linkage,
w with a partial double-bond character.
n this bond not freely rotating:
w 6 atoms constrained to a plane:
Ci, Ci, Oi, Ni+1,Hi+1,Ci+1.
The peptide bond can adopt one of 2
configurations:
n based on the positions of Ci and Ci+1.
o
n The trans-configuration (i= 180 ):
w energetically favored…usually adopted.
n The cis-configuration: (i= 0o):
w sterically hindered…energetically unfavorable.
w exception: Proline (cis and trans nearly
The Peptide Bond (cont.)
Fixing the peptide bond in either cis or trans:
n leaves the backbone with only two degrees of freedom
at Ri.
n these are expressed by two torsion angles:
w  = angle about the Ni-Ci bond.
n defined by Ci-1-Ni-Ci.-Ci.
w  = angle about the Ci-Ci bond.
n defined by Ni-Ci-Ci-Ni+1.
n bond lengths are nearly constant…therefore:
The Ramachandran Plot
The Ni-Ci bond, and Ci-Ci bond are single bonds:
n in principle, each may rotate freely…
w  and could then assume any values b/w +/- 180o.
w however, this is true only for Glycine (R = H).
For other R-groups, non-covalent interactions b/w
adjacent side-chains:
n place energetic constraints on  and .
w thus, some conformations (,) are sterically disallowed.
Sasisekharan and Ramachandran (1968):
n first plotted the van der Waals energies of interaction vs.
(,).
w using poly-L-Alanine
n R = Me, the minimally constrained group (with a C-Carbon).
w with the trans-configuration for each peptide bond.
n the resulting plot is a ‘Ramachandran Plot’.
The Ramachandran Plot
(cont.)
Plot shown in terms of allowed regions…
w here, c = helical repeat, with +/- = right/left-handed.
n dark regions = sterically allowed.
n lightly shaded regions = moderately disfavored.
n others = disallowed.
Torsion angles of helices:
n all in allowed regions.
n Note: L- left handed -helix.
w Glycine only (achiral).
Not a good model for:
n Glycine or Proline.
n residues w/ bulky side-chains.
Conclusion
In this Lecture, we have discussed:
n Amino Acid Residues
w Characteristics and Hydrophobicities
o
n Protein 2 structure:
w The local, helical structures adopted by polypeptides.

We will continue our discussion in Lecture 4:

n The intermediate level structure of Proteins:
w Super-2o and Domainal structure of Proteins.
n Methods of Visualization of the overall 3-D structures
of Proteins.
n Protein 3o structure:
w contact plots.
n Protein 4o structure.