(J-374) Structural Characterization of Lipase using Small-angle X-ray scattering and Quartz Crystal Microbalance

Polymer-protein hybrids have attracted growing interest in the fields of biomedical engineering  and biochemistry. By complexing a protein with a polymer, it is possible to improve the stability of the  protein under otherwise denaturing conditions.  We are designing these polymers by using machine-learning approaches as well as by understanding the specific interactions between enzymes and synthetic random copolymers whose compositions can be adjusted based on the surface characteristics of the protein. To properly understand the interaction between lipase, and random copolymers, small-angle X-ray scattering  (SAXS) was used to characterize the physical structure of lipase and the formed complexes. Quartz-crystal microbalance with dissipation (QCMD)  was used to understand the complex formation when an enzyme is immobilized.

SAXS experiments were done at the National Synchrotron Light Source II (NSLS-II) of Brookhaven National Laboratory (Upton, NY). The data (q range of 0.001-0.5 Å-1). Data were analyzed using BioXTAS RAW after subtracting the buffer scattering. The radius of gyration (Rg) was calculated by Guinier analysis, flexibility from the Kratky plots, and the shape information from the  pair-distance distribution functions, P(r). QCMD measurements were carried out on gold sensors using an Omega instrument (Biolin Scientific).

Guinier analysis of the data showed our sample to have an Rg ~50 Å (Figure 1A) much greater than the 29Å expected  for a 33 k Da lipase enzyme.  More importantly, the Kratky plot (Figure 1B) showed the molecule to be disordered, which in fact lipase is a globular, compact molecule.  The  P(r) function showed our molecule to have a maximum dimension of 120 Å rather than 50 Å  expected of a compact lipase. These data prompted us to carry out further measurement with light scattering and differential scanning fluorimetry (Figure 1C).  These measurements showed that the sample had only 1% of lipase. The excipient that was used had a size that overlapped that of the enzyme and hence not distinguishable.  Despite the low concentration of the enzyme, we were able to measure the activity of the enzyme even when it was immobilized in the QCMD crystals.  We were able to see differences between the high- and low-performing polymer complexed with the enzyme.