  One of the most common mutations in the serine protease inhibitor Kazal type
1 (SPINK1) gene is the N34S variant which is strongly associated with chronic
pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk
factor, the underlying pathologic mechanism is still unknown. In the present
study, we investigated the impact of physiological stress factors on SPINK1
protein structure and trypsin inhibitor function using biophysical methods. Our
circular dichroism spectroscopy data revealed differences in the secondary
structure of SPINK1 and N34S mutant suggesting protein structural changes
induced by the mutation as an impairment that could be disease-relevant. We
further confirmed that both SPINK1 (KD of 0.15 +/- 0.06 nM) and its N34S
variant (KD of 0.08 +/- 0.02 nM) have similar binding affinity and inhibitory
effect towards trypsin as shown by surface plasmon resonance and trypsin
inhibition assay studies, respectively. We found that stress conditions such as
altered ion concentrations (i.e. potassium, calcium), temperature shifts, as
well as environmental pH lead to insignificant differences in trypsin
inhibition between SPINK1 and N34S mutant. However, we have shown that the
environmental pH induces structural changes in both SPINK1 constructs in a
different manner. Our findings suggest protein structural changes in the N34S
variant as an impairment of SPINK1 and environmental pH shift as a trigger that
could play a role in disease progression of pancreatitis.
