section
4.7
Protein Folding and Associated Diseases
59
of the changes in properties that may be caused by denat-
uration are as follows:
1
. decreased solubility (often but not invariably);
2
. alteration in the internal structure and arrangement of
peptide chains that does not involve breaking the
peptide bonds (e.g., separation of subunits of
oligomeric proteins);
3. disrupted secondary structure (e.g., loss of helical
structure);
4. increased chemical reactivity of functional groups of
amino acids, particularly ionizable and sulfhydryl
groups (e.g., shift of pK values);
5. increased susceptibility to hydrolysis by proteolytic
enzymes;
6
. decrease or total loss of the original biological
activity; and
7. loss of crystallizability.
Studies by Anfinsen of the reversible denaturation of the
pancreatic enzyme ribonuclease prompted the hypothesis
that secondary and tertiary structures are derived inclu-
sively from the primary structure of a protein (Figures 4-11
and 4-12). RNase A, which consists of a single polypep-
tide chain of 124 amino acid residues, has four disulfide
bonds. Treatment of the enzyme with
8
M urea, which dis-
rupts noncovalent bonds, and /f-mcrcaptoethanol, which
reduces disulfide linkages to cysteinyl residues, yields a
random coil conformation.
However, if both reagents are removed and the cys-
teinyl residues are allowed to oxidize and re-form disul-
fide bonds, of the 105 different possible intramolecular
10
FIGURE 4-11
Amino acid sequence of bovine ribonuclease A. The molecule contains
four disulfide bridges.
Enzyme with
native confor-
mation and
full enzyme
activity
Denaturation
72,
SH
___
;
SH
Reduced and
Ï& J
/ SH
/H S
denatured
,„H S
Η
*110
loss of activity
( 1
) Removal of urea and
P-mercaptoethanol
(2) Oxidation in air
Renaturation
Return to native
conformation and
full activity
FIGURE 4-12
Denaturation and renaturation of ribonuclease A.
combinations of disulfide linkages, only the four correct
bonds form, and the denatured enzyme returns to its orig-
inal, biologically active structure (Figure 4-12). These ex-
periments are taken as proof that the primary structure
(which is genetically controlled) determines the unique
three-dimensional structure of a protein. However, as de-
scribed below, it is now known that the folding of some
proteins is assisted by other proteins.
4.7 Protein Folding and Associated Diseases
Proteins are synthesized on ribosomes as nascent polypep-
tides in the lumen of the endoplasmic reticulum (ER). The
amino acid sequence of proteins that determines the sec-
ondary and tertiary structures is dictated by the nucleotide
sequence of mRNA. In turn, mRNA sequences are deter-
mined by DNA sequences (Chapters 23-25). As discussed
earlier, the classic experiments of Pauling and Anfinsen
led to the concepts that certain key amino acids at the
proper positions are essential for the folding of proteins
into a three-dimensional, functional, unique conformation.
It is amazing that, of hundreds of millions of conforma-
tional possibilities, only a single conformational form is
