Protein Denaturation and Folding:
A native protein conformation is only marginally stable. In addition, most
proteins must maintain conformational flexibility to function. The continual
maintenance of the active set of cellular proteins required under a given set of
conditions is called proteostasis. Cellular proteostasis requires the coordinated
function of pathways for protein synthesis and folding, the refolding of proteins
that are partially folded, and the sequestration and degradation of proteins that
have been irreversibly unfolded.
The marginal stability of most proteins can produce a tenuous balance between
folded and unfolded states. As proteins are synthesized on ribosomes, they must
fold into their native conformations. Sometimes this occurs spontaneously, but
more often it occurs with the assistance of specialized enzymes and complexes
called chaperones. Many of these same folding helpers function to refold
proteins that become transiently unfolded. Proteins that are not properly folded
often have exposed hydrophobic surfaces that render them ‘sticky’, leading to
the formation of inactive aggregates. These aggregates may lack their normal
function but are not inert. All cells have elaborate pathways for recycling and/or
degrading proteins that are irreversibly misfolded.
Loss of Protein Structure Results in Loss of Function:
Conditions different from those in the cell can result in protein structural
changes, large and small. A loss of 3D structure sufficient to cause loss of
function is called denaturation. The denatured state does not necessarily equate
with complete unfolding of the protein and randomization of conformation.
Under most conditions, denatured proteins exist in a set of partially folded states.
Most proteins can be denatured by heat, which has complex effects on many
weak interactions in a protein (primarily on H-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.
The abruptness of the change suggests that unfolding is a cooperative process:
loss of structure in one part of the protein destabilizes other parts.
Proteins can also be denatured by extremes of pH, by certain miscible organic
solvents such as alcohol or acetone. Each of these denaturing agents represents a
relatively mild treatment in the sense that no covalent bonds in the polypeptide
chain are broken. Organic solvents, urea, and detergents act primarily by
disrupting the hydrophobic interactions that make up the stable core of globular
proteins; urea also disrupts hydrogen bond; extremes of pH alter the net charge
on the protein, causing electrostatic repulsion and the disruption of some
hydrogen bonding. The denatured structures resulting from these various
treatments are not necessarily the same.
Denaturation often leads to protein precipitation, a consequence of protein
aggregate formation as exposed hydrophobic surfaces associate. The aggregates
are often highly disordered. Amino Acid Sequence Determines Tertiary Structure:
Denaturation of some proteins is reversible. Certain globular proteins denatured
by heat, extremes of pH, or denaturing reagents 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 is the denaturation and renaturation of ribonuclease. Purified
ribonuclease A denatures completely in 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 is accompanied by a 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 restorating of its catalytic activity. The refolding of ribonuclease is so
accurate that the four intrachain disulfide bonds are re-formed in the same
positions in the renatured molecule as in the native ribonuclease.
The Anfinsen experiment 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, 3D structure. Subsequent work has shown that only a
minority of proteins, many of them small and inherently stable, will fold
spontaneously into their native form. Even though all protein