Introduction about protein structure

• The spatial arrangement of atoms in a protein or any part of a protein is called its conformation.
• A change in protein conformation could occur by rotation about single bonds without breaking it.
• Of the many conformations that are theoretically possible in a protein containing hundreds of single bonds, only a few
generally predominate under biological conditions.
• Why there are multiple stable conformations of proteins? Because most proteins bind to other molecules or catalyze
reactions.
• A Protein’s Conformation Is Stabilized Largely by Weak Interactions.
• Many proteins do not have disulfide bonds because the environment within most cells is highly reducing due to high
concentrations of reductants such as glutathione, and most sulfhydryl groups will thus remain in the reduced state.
• Outside the cell, the environment is often more oxidizing, and disulfide formation is more likely to occur.
• In eukaryotes, disulfide bonds are found primarily in extracellular proteins.
• Disulfide bonds are also uncommon in bacterial proteins. However, thermophilic bacteria, as well as the archaea, typically
have many proteins with disulfide bonds to stabilize proteins. This is presumably an adaptation to life at high temperatures.
For all proteins of all organisms, weak interactions are especially important in the folding of polypeptide chains into
their secondary and tertiary structures. The association of multiple polypeptides to form quaternary structures also
relies on these weak interactions.
 Peptide bond is rigid and planar
• In the late 1930s, Linus Pauling and Robert Corey performed a series of studies
to understand the protein structure. They began with a careful analysis of the
peptide bond.
• The α carbons of adjacent amino acid residues are separated by three covalent
bonds, arranged as Cα—C—N—Cα
• X-ray diffraction studies showed that the peptide C—N bond is somewhat
shorter than the C—N bond in a simple amine.
• This indicated partial sharing of two pairs of electrons between the carbonyl
oxygen and the amide nitrogen, setting up a small electric dipole.
• The six atoms of the peptide group lie in a single plane.
• From these findings Pauling and Corey concluded that the peptide C—N bonds,
because of their partial double-bond character, cannot rotate freely. Rotation is
permitted about the N—Cα and the Cα—C bonds. The backbone of a
polypeptide chain can thus be pictured as a series of rigid planes, with
consecutive planes sharing a common point of rotation at Cα. The rigid peptide
bonds limit the range of conformations possible for a polypeptide chain.
• Other single bonds in the backbone may also be
rotationally hindered, depending on the size and
charge of the R groups.
• In principle, phi and psi can have any value between
-180º and +180º but many values are prohibited by
steric interference between atoms in the polypeptide
backbone and amino acid side chains.
• Allowed values for phi and psi are graphically
revealed when psi is plotted versus phi in a
Ramachandran plot (as shown in figure) introduced
by G. N. Ramachandran.
The third level of structure found in proteins, tertiary structure, refers to the spatial arrangement of amino acids
that are far apart in the linear sequence as well as those residues that are adjacent. Once folded, the three-dimensional,
biologically active (native) conformation of the protein is maintained not only by hydrophobic interactions, but also by
electrostatic forces, hydrogen bonding and, if present, the covalent disulfide bonds.

Proteins containing more than one polypeptide chain, such as hemoglobin (shown below), exhibit a fourth level of protein
structure called quaternary structure. This level of structure refers to the spatial arrangement of the polypeptide
subunits and the nature of the interactions between them. These interactions may be covalent links (e.g. disulfide bonds) or
noncovalent interactions (electrostatic forces, hydrogen bonding, hydrophobic interactions).

Residues (%)
Protein stability
Most proteins must maintain conformational flexibility to

The native three-dimensional conformation function. The continual maintenance of cellular proteins is

of a protein is maintained by a range of called proteostasis. Cellular proteostasis requires the

coordinated function of pathways; protein synthesis, folding, the
noncovalent interactions (electrostatic
refolding of proteins that are partially unfolded, and the
forces, hydrogen bonds, hydrophobic
sequestration and degradation of proteins that have been
forces) and covalent interactions (disulfide
irreversibly unfolded or are no longer needed. In all cells, these
bonds), in addition to the peptide bonds networks involve hundreds of enzymes and specialized proteins.
between individual amino acids.

• Electrostatic forces
• van der Waals forces
• Hydrogen bonds
• Hydrophobic forces
• Disulfide bonds
Pathways that contribute to proteostasis
Three kinds of processes contribute to proteostasis, in some
cases with multiple contributing pathways.
First, proteins are synthesized on a ribosome.
Second, multiple pathways contribute to protein folding, many
of which involve the activity of complexes called chaperones.
Chaperones also contribute to the refolding of proteins that are
partially and transiently unfolded.
Finally, proteins that are irreversibly unfolded are subject to
sequestration and degradation by several additional pathways.
Partially unfolded proteins and protein-folding intermediates
that escape the quality control activities of the chaperones and
degradative pathways may aggregate, forming both disordered
aggregates and ordered amyloid like aggregates that
contribute to disease and aging processes.
The UPR (unfolded protein response) is
activated in response to an accumulation of
unfolded or misfolded proteins in the lumen of the
endoplasmic reticulum. In this scenario, the UPR
has three aims: initially to restore normal function
of the cell by halting protein translation,
degrading misfolded proteins, and activating the
signalling pathways that lead to increasing the
production of molecular chaperones involved
in protein folding. If these objectives are not
achieved within a certain time span or the
disruption is prolonged, the UPR aims
towards apoptosis.
 Protein Denaturation and Folding
• Protein structures have evolved to function in particular cellular environments.
• Conditions different from those in the cell can result in protein structural changes, large and small. A
loss of three-dimensional structure sufficient to cause loss of function is called denaturation.
• Most proteins can be denatured by heat, which affects the weak interactions in a protein (primarily
hydrogen bonds. If the temperature is increased slowly, a protein’s conformation generally remains
intact until an abrupt loss of structure (and function) occurs over a narrow temperature range,
suggests that unfolding is a cooperative process: loss of structure in one part of the protein
destabilizes other parts.
• The very heat-stable proteins of thermophilic bacteria have evolved to function at the temperature of
hot springs (~100º C).
• Proteins can be denatured not only by heat but by extremes of pH, by certain miscible organic
solvents such as alcohol or acetone, by certain solutes such as urea and guanidine hydrochloride, or
by detergents.
• The covalent bonds in the polypeptide chain are broken by these denaturing agents. Organic
solvents, urea, and detergents act primarily by disrupting the hydrophobic interactions that make up
the stable core of globular proteins; extremes of pH alter the net charge on the protein, causing
electrostatic repulsion and the disruption of some hydrogen bonding. The denatured states obtained
with these various treatments need not be equivalent.
Amino Acid Sequence Determines Tertiary
Structure
The most important proof of this came from experiments showing that denaturation of
some proteins is reversible. Certain globular proteins denatured will regain their native
structure and their biological activity if returned to conditions in which the native
conformation is stable. This process is called renaturation.
A classic example of denaturation and renaturation: Purified ribonuclease can be
completely denatured by exposure to a concentrated urea solution in the presence of a
reducing agent. The reducing agent cleaves the four disulfide bonds to yield eight Cys
residues, and the urea disrupts the stabilizing hydrophobic interactions, thus freeing the
entire polypeptide from its folded conformation. Denaturation of ribonuclease by urea
causes complete loss of catalytic activity.
When the urea and the reducing agent are removed, the randomly coiled, denatured
ribonuclease spontaneously refolds into its correct tertiary structure, with full
Renaturation of unfolded, denatured ribonuclease.
restoration of its catalytic activity. The refolding of ribonuclease is so accurate that the Urea is used to denature ribonuclease, and
mercaptoethanol to reduce and thus cleave the
four intrachain disulfide bonds are re-formed in the same positions in the renatured disulfide bonds to yield eight Cys residues.
Renaturation involves reestablishment of the correct
molecule as in the native ribonuclease. disulfide cross-links.
weak bonding interactions are required for correct positioning
of disulfide bonds and assumption of the native conformation

Mathematically, the eight Cys residues could recombine at random to form up to four disulfide
bonds in 105 different ways.
In fact, the random distribution of disulfide bonds is obtained when the disulfides are allowed
to reform in the presence of denaturant, indicating that weak bonding interactions are required
for correct positioning of disulfide bonds and assumption of the native conformation.

Conclusion: This classic experiment, carried out by Christian Anfinsen in the 1950s, provided
the first evidence that the amino acid sequence of a polypeptide chain contains all the
information required to fold the chain into its native, three-dimensional structure.
Later, similar results were obtained using chemically synthesized, catalytically active
ribonuclease. This eliminated the possibility that some minor contaminant in Anfinsen’s
purified ribonuclease preparation might have contributed to the renaturation of the enzyme.
Some Proteins Undergo Assisted Folding
Not all proteins fold spontaneously as they are synthesized in the cell.
Folding for many proteins requires chaperones, proteins that interact with
partially folded or improperly folded polypeptides, facilitating correct
folding pathways or providing microenvironments in which folding can
occur. Several types of molecular chaperones are found in organisms
ranging from bacteria to humans.
Two major families of chaperones are the Hsp70 and the chaperonins.
The Hsp70 family of proteins are more abundant in cells stressed by
elevated temperatures. These chaperones “protect” both proteins subject to
denaturation by heat and new peptide molecules being synthesized (and
not yet folded). Hsp70 proteins also block the folding of certain proteins
that must remain unfolded until they have been translocated across a
membrane. Some chaperones also facilitate the quaternary assembly of
oligomeric proteins.
The folding pathways of some proteins require two enzymes that
catalyze isomerization reactions.
• Protein disulfide isomerase (PDI) is a widely distributed enzyme
that catalyzes the interchange, or shuffling, of disulfide bonds until
the bonds of the native conformation are formed. Among its
functions, PDI catalyzes the elimination of folding intermediates
with inappropriate disulfide cross-links.

• Peptide prolyl cis-trans isomerase (PPI) catalyzes the

interconversion of the cis and trans isomers of peptide bonds formed
by Pro residues, which can be a slow step in the folding of proteins
that contain some Pro peptide bonds in the cis configuration.
Defects in Protein Folding causes diseases
Despite the many processes that assist in protein folding,
misfolding does occur. In fact, protein misfolding is a
substantial problem in all cells, and a quarter or more of all
polypeptides synthesized may be destroyed because they do
not fold correctly. In some cases, the misfolding contributes
to the development of serious disease.
Many conditions, including type 2 diabetes, Alzheimer
disease, Huntington disease, and Parkinson disease, are
associated with a misfolding mechanism: a soluble protein
that is normally secreted from the cell is secreted in a
misfolded state and converted into an insoluble extracellular
amyloid fiber with a diameter of 7 to 10 nm. The diseases
are collectively referred to as amyloidoses.