refolding into native shape. (2) The RNase solution was diluted during dialysis by the water entering the dialyzer by osmosis. The clear
zones would become larger and the results more obvious with a longer incubation time. The clear zone at D is as large as that at A, indicating that boiling had no effect on the activity of RNase because its
four disulfide bonds strongly hold the conformation of the molecule.
The enzyme RNase can spontaneously refold into its native, three-dimensional shape after being fully unfolded in vitro, which is consistent with the prediction derived from hypothesis 1. Since the
unfolded RNase has lost all the information of its shape imparted
from the process of protein synthesis, while the only information
left is the amino acid sequence, we can conclude that the folding
of RNase is determined by its amino acid sequence.
The above investigation only shows that protein can refold into its
native shape spontaneously, but it offers no explanation for that.
For AP or other advanced biology courses, further investigation
can be conducted to test the thermodynamic hypothesis of Anfinsen.
In AP biology, students are required to know “the specific order of
amino acids in a polypeptide (primary structure) interacts with the
environment to determine the overall shape of the protein . . . chemical properties (hydrophobic, hydrophilic and ionic), and the interactions of these R groups determine structure and function of that
region of the protein” (College Board, 2015, 4.A.1.a.2). The thermodynamic hypothesis stipulates that the native shape of a protein is
dependent on the interactions of the R groups with the environment
to reach the thermodynamically lowest energy state. This state will
change when the external environment changes, such as in the presence of urea. This can be tested by placing the dialyzer in urea solution so that only 2-ME is removed. When the unfolded RNase
refolds in the presence of urea, it will refold into many “scrambled”
proteins as a result of the randomly formed disulfide bonds. These
scrambled molecules cannot refold into native shape even after urea
is removed. However, when some 2-ME is added to break the randomly formed disulfide bonds, the RNase can restore its activity by
refolding to native shape (Figure 1).
The instructional approach employed is guided inquiry (Buck et al.,
2008), where the question is provided and the experimental design
is partly done by students under teacher guidance. The experiment
assumes students have a basic understanding of DNA, protein struc-
ture, protein synthesis, enzymes, and dialysis. Students were first
told that they would replicate a Nobel-winning experiment to tackle
an important question in biology—how a polypeptide chain folds
into three-dimensional shape to become a functional protein. After
reviewing the relevant concepts, students in groups of four were
asked to design an experiment to test the two hypotheses. Students
had no difficulty in understanding how to measure RNase activity
with RNA agar since they had done similar experiment with amylase
and starch agar. After group discussion, each group presented their
designs to the class. Their designs mainly differed on how to do rep-
lication and control on the RNA agar plate. All groups had proposed
a control with only buffer and denaturants, while two groups men-
tioned a control with boiled RNase. We did not point out that RNase
is heat resistant and deliberately used it as a discrepant event to con-
front their misconception that all enzymes can be denatured by heat.
The discussion was guided by the teachers, but no “correct” answers
were provided. During the incubation of the agar plates that took 1
to 1.5 hours, students could have a lunch break or write up part of
their lab report. After incubation, each group showed its plates to the
class with a visualizer and explained the results. The whole experi-
ment was finished within 2.5 hours, excluding the time for incuba-
tion. Alternatively, the experiment can be done in two consecutive
days: On the first day, students investigate the RNase activity with
RNA agar, and then do dialysis themselves to test for the hypotheses.
After dialysis overnight, they test the activity of the dialyzed RNase
solution on the second day.
In general, most students were excited and engaged by the
experiment because they felt that they were conducting a Nobel-
winning experiment to discover an important biological principle.
Particularly important is that they found a Nobel-winning experi-
ment to be within their grasp, which can bolster their self-confidence
in learning science. The openness of the investigation is also key to
engaging the students because they are often asked to follow pre-
scribed procedures to verify known facts in ordinary school experi-
ments. Despite the fact that the students are not academically
strong and the concept of protein folding is unfamiliar to them, their
lab reports show that they understand the experiment well and can
explain the results satisfactorily. The overall performance of the
students has supported that even complex, college-level experi-
ments, when properly guided and designed, can be conducted in
secondary classrooms for learning important biological principles
and science practices.
AAAS Project 2061 (n.d.) [Pilot and field test data collected between 2006
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