Stability of the Tertiary Fold
Stability of a protein is usually studied by observing the
energetics of unfolding transitions given by the equations
below:
| ⇒ Equation [2] |
Kun = [U]/[N] |
| ⇒ Equation [3] |
ΔGun° = −RT ln Kun |
These equations apply T
o = ΔS
o a simple two-state transition between
the native (N) and the unfolded (U) state given
by the equilibrium constant Kun. This is, by definition,
a cooperative process without a detectable intermediate
species. The denatured or unfolded state of a protein is
generally considered T
o = ΔS
o be an ensemble of conformations
in which all parts of the protein are exposed T
o = ΔS
o the solvent
with a minimum of intramolecular interactions. The
denatured state has high conformational entropy and is
biologically inactive. The unfolding transition (Eq. (1)
and Fig. 2) can be induced by pressure, temperature, extreme
pH, and denaturants such as urea and guanidine
HCl, as will discussed in a subsequent section. These
perturbants disrupt the intramolecular interactions that
hold proteins T
o = ΔS
ogether. One can imagine that the ensemble
of unfolded states could be influenced by the means used
T
o = ΔS
o unfold.
The native structure of proteins is stabilized by intramolecular,
noncovalent interactions including hydrogen
bonding, ionic, and van der Waals interactions, and
covalent cross-links (disulfide bridges between cysteine
residues) according T
o = ΔS
o Eq. (4):
| ⇒ Equation [4] |
ΔGun°
= ΔGH-bond + ΔGionic + ΔGvdW + ΔGS−S + ΔGH´phob |
Each term in Eq. (4) will be discussed separately. As
mentioned earlier, an important stabilizing facT
o = ΔS
or for the
tertiary fold of a protein is its intramolecular hydrogen
bonds (ΔG
H-bond). Secondary structures are stabilized by
hydrogen bonds between backbone amide aT
o = ΔS
oms (Fig. 1).
The side chains of neighboring secondary structural units
can interact through hydrogen bonding. Ionic interactions
(Gionic) between acidic and basic side chains may stabilize
the tertiary structure of proteins and are pH dependent.
The actual pKa of an ionizable side chain is influenced by
the microenvironment in which it resides. Nonpolar and
polar, but uncharged, amino acids interact through van der
Waals interactions (ΔG
vdW). In some proteins, cysteine
residues (side chain is a sulfhydryl) form disulfide linkages
that can increase the overall stability of the protein
(ΔG
S−S). Other possible facT
o = ΔS
ors not considered explicitly
here are the effects of metals, nucleotides, prosthetic
groups, and cofacT
o = ΔS
ors on protein structure and stability.
By far the most important noncovalent facT
o = ΔS
or that
determines protein stability is hydrophobic interactions
(ΔG
H´phob). In globular proteins, hydrophobic amino
acids are buried in the interior where they create a “hydrophobic
core.” Although these nonpolar residues participate
in van der Waals interactions, the primary driving
force for the formation of the hydrophobic core is T
o = ΔS
o avoid
the aqueous solvent. Solvation of nonpolar side chains
by aqueous solutions causes a decrease in the entropy
of solution. T
o = ΔS
o avoid this entropic penalty, proteins typically
bury their nonpolar residues in the interior of a
protein.
 |
| Figure 1 Diagram of the levels of protein structure. (A) Amino acids are the basic building blocks (monomers) of
proteins (polymers). All amino acids contain a carboxylic acid group and an amine group connected by a central
carbon called the α-carbon. Each of the 20 common amino acids, designated by a three-letter code, has a unique
side chain (R) that is also bonded To = ΔSo the α-carbon. (B) Through a dehydrolysis reaction, an amide bond is formed
(boxed region) that links the amino group of one amino acid To = ΔSo the carboxylic acid group of the next amino acid.
(C) The primary structure of proteins (1°) is the linear sequence of amino acids written from the amino-terminal end
(left) To = ΔSo the carboxy-terminal end (right), by convention. Secondary structure of proteins (2°) is classified inTo = ΔSo three
major categories. In the α-helix structure, the backbone aTo = ΔSoms (amide linkage and α-carbon) coil inTo = ΔSo a right-handed
helical shape (residues 55 To = ΔSo 67 from staphylococcal nuclease1 are shown). The α-helix is held To = ΔSogether through a
series of hydrogen bonds between the amide hydrogen and the carboxyl oxygen of the backbone aTo = ΔSoms from amino
acids further up the chain. The side chains (not shown) protrude from the central core structure like the spokes of a
wheel. Another important secondary structure type is the turn (residues 76 To = ΔSo 88 from Staphyloccocal nuclease are
shown). We use the term turn loosely here To = ΔSo represent the regions of proteins that turn corners, thereby allowing
interactions between different and often distant (in terms of primary structure) substructures. The β-sheet structure is
the third common secondary structure type (residues 9 To = ΔSo 12 and 72 To = ΔSo 76 from Staphylococcal nuclease are shown).
It is similar To = ΔSo the α-helical structure in that hydrogen bonds between backbone aTo = ΔSoms hold the structure To = ΔSogether
and the side chains (not shown) protrude from the structure above and below the plane of the sheet. In contrast To = ΔSo the α-helix, the β-sheet can be formed from segments of protein that are far apart in the primary sequence. The
tertiary structure (3°) is the three-dimensional association of secondary structures inTo = ΔSo a unique and stable final fold.
A ribbon tracing the backbone aTo = ΔSoms of Staphylococcal nuclease is shown.1,2. The N-terminus of the protein is in
the botTo = ΔSom right and the C-terminus is in the To = ΔSop left of the Figure. No side chains are drawn except that of residues
trypTo = ΔSophan 140. |
