These concepts also give a good example on how biological
structure is related to function, a crosscutting concept of NGSS.
Nature of Science
This investigation can be used to teach about many aspects of the
nature of science. Enzyme refolding into its native shape is not
directly observed but inferred from the observation that the enzyme
regains its activity after denaturation. There could be alternative
hypotheses that account for this observation: 2-ME and urea may
act as enzyme inhibitors to alter the shape of the active site rather
than unfold the protein completely; or, the RNase may not have
refolded into its native shape but another shape that happens to have
the same catalytic function as the native one. This poses the problem
of underdetermination between scientific theory and evidence
(Duhem, 1954). Nonetheless, these alternative hypotheses had been
ruled out by further empirical evidence obtained from X-ray diffraction, UV spectrophotometry, chromatography, and electrophoresis
that the structures and shapes of RNase do change during unfolding
and refolding (White, 1961). Therefore, despite that scientific theory
is inference and thus inherently tentative, it is nonetheless reliable in
light of its multiple lines of empirical evidence.
The original question Anfinsen and his colleagues wanted to
answer was not protein folding, but the catalytic activity of RNase.
They cleaved the disulfide bonds of RNase with the aim of finding
out if the bonds were related to its catalytic function. But accidentally,
they found that RNase unfolded and refolded, opening up the
research area of protein folding (Sela et al., 1957; White, 1961). This
shows that scientific inquiry may not be a logical and linear process
starting from a definite question. Moreover, Anfinsen could not have
envisaged that his discovery about protein folding would help understand cystic fibrosis, Alzheimer’s disease, and mad cow disease; the
significance of a scientific discovery is often not obvious at the start.
In 1989, Anfinsen commented that his own work in 1955 represents a “beautiful example of how an entirely acceptable conclusion
can be reached that is entirely wrong because of the paucity of knowledge at that particular time” (Anfinsen, 1989). In his 1955 paper
(Anfinsen et al., 1955), Anfinsen concluded that an ordered shape
of a protein is not needed for its catalytic function. This is a wrong
but reasonable conclusion at the time because protein sequencing
and crystallography were not available. Instead of regarding it as
“fraud” in science, Anfinsen argued that the advance of science
requires “ongoing refinement of data . . . and reinterpretation of formerly held ‘truth’.” He himself had demonstrated this self-correcting
nature of science by spending “the following 15 years or so completely
disproving the conclusions.” Even Anfinsen’ s Nobel-winning discovery has since been found not completely correct: protein folding is not
solely determined by amino acid sequence but modified by small proteins called chaperones in cells (Ellis, 1987). Prion, the infectious protein of mad cow disease, is a kind of chaperone.
For decades, protein folding has been an active field of research in
biology, chemistry, biochemistry, computer science, and physics. The
mystery of protein folding, however, has not been cracked completely
after over 50 years. The intramolecular and intermolecular interactions of a protein during folding are hugely complex, and it is still
not possible to predict the exact shape of a protein according to its
amino acid sequence. This again shows that scientific inquiry is an
ongoing process of problem solving where solving one question will
open up many more questions.
Adaptation of Anfinsen’s Experiment
To make Anfinsen’s protein folding experiment (Anfinsen & Haber,
1961) doable in secondary laboratory, some procedures like spectrophotometry, gel chromatography, and gel filtration are replaced by
RNA agar to show the ribonuclease activity and dialysis to remove
the denaturants. The design principles of the experiment make reference to a study on refolding human serum albumin (Burton et al.,
1989). Apart from materials, another consideration is whether secondary students understand the concepts to understand the experiment. A senior secondary biology student should understand the
concepts of gene expression, protein structure and functions, and
enzymes, but the concepts of thermodynamics and hydrophobic
interactions of the polypeptides seem beyond reach of some students, particularly those not studying advanced chemistry and physics. Therefore, we design a basic version of the experiment that only
touches upon protein unfolding and refolding, and a more advanced
version that goes further to test for the thermodynamic hypothesis.
Protein Folding Investigation
Anfinsen’s key experiment in 1961 (Anfinsen & Haber, 1961) was
modified into a biology investigation, which had been tried out on
23 secondary students from five schools at a university in Hong
Kong. The detailed procedure and preparation of the experiments
can be found in Online Supplemental Material.
A protein functions only when it is in a specific three-dimensional
shape. What determines the folding of a polypeptide chain into its
native, three-dimensional shape?
1. Protein folding is wholly determined by the amino acid
sequence of the polypeptide chain itself.
2. Protein is folded into a specific shape by some instructions
other than its amino acid sequence in the cytoplasm, such
as tRNA, ribosome, or enzymes.
According to hypothesis 1, a fully unfolded protein can spontaneously refold back into its native shape in vitro and regain its catalytic function. This is because the instruction required for correct
refolding is already encoded in the amino acid sequence of the
polypeptide itself. According to hypothesis 2, however, a fully
unfolded protein will not spontaneously refold back into its native
shape in vitro because the refolding requires the protein synthesis
machinery and other instructions in cytoplasm.
Design of the Investigation
The unfolding and refolding of an enzyme ribonuclease (RNase) is
studied. The activity of RNase can be shown by RNA agar plate,
where a clear zone is produced after RNA is broken down by the
RNase. To unfold the RNase, 2-mercaptoethanol (2-ME) and urea
are used as denaturants. 2-ME breaks down the four disulfide bonds
of RNase, urea disrupts the polar nature of the solvent, and together