Reports: DNI449968-DNI4: NMR Study of Kinetics of Protein/Cellulose Interactions

Junji Iwahara, PhD , University of Texas Medical Branch

As society’s demand for sustainable energy is increasing, efficient production of biofuels from biomass is becoming more important than ever. In order to increase the efficiency via knowledge-based engineering of cellulase enzymes that digest cellulose, it is crucial to understand the kinetics and mechanisms of the protein / cellulose interactions. The main focus in this project was on the kinetics of protein-cellulose interactions involving the cellulose-binding domain (CBD) of the cellulase enzyme 9B from Cellulomonas fimi cellulase  (Cel9B). The CBD bind noncrystalline cellulose and cello-oligosaccharides, and play important role to keep the enzymes in close proximity from the substrate. By using NMR spectroscopy, we have successfully achieved our two Specific Aims: 1) analysis of the kinetics of protein translocation between cello-oligosaccharide molecules, and 2) determination of the kinetic rate constants for protein / cello-oligosaccharide dissociation and association.

For Specific Aim 1, we studied the kinetic mechanisms for exchange between the cellohexaose- and cellotetraose-bound forms of the Cel9B CBD.  Using 2H/15N-labeled proteins, we analyzed NMR line-shapes in 1H-15N TROSY spectra recorded for the three samples: 1) the CBD/cellohexaose complex, 2) CBD/cellotetraose complex, and 3) mixtures of the CBD/cellohaxose and CBD/cellotetraose complexes. From these NMR data, we determined the pseudo-first-order rate constants for the exchange between cellohexaose- and cellotetraose-bound forms of the Cel9B CBD. The exchange can occur via two mechanisms: 1) dissociation followed by re-association, and 2) direct transfer mediated by collisions between a complex and a free ligand. For the “dissociation & re-association” mechanism, the rate-limiting step is dissociation if concentration of ligand [L] is much higher than the dissociation constant Kd (Note that this condition leads to kon[L] >> koff). In this limit, the kinetic rates should be virtually independent of [L], because dissociation is a first order process. On the other hand, direct transfer is a single-step second-order process, and therefore its rates are proportional to [L]. By measuring the pseudo-first-order rate constants for the exchange between cellohexaose- and cellotetraose-bound forms at some different concentrations of free ligands, we have determined the second-order rate constants for direct transfer. They were 2.9 x 106 M-1 s-1 for direct transfer from the cellotetraose-bound state to the cellohexaose-bound state and 3.9 x 105 M-1 s-1 for the reverse process. These kinetic data suggest that the cellulase enzyme can rapidly transfer from one substrate to another via direct transfer on noncrystalline.

For Specific Aim 2, we studied the association / dissociation kinetics for the protein-cellulose interactions by using the 15N CPMG relaxation dispersion and NMR line-shape analysis methods. We have found that the Cel9B CBD in the free state undergoes dynamic transitions between two states Pa (75%) and Pb (25%) with an exchange rate (kex) of 120 s-1. By applying the McConnell equations for three-state models, we analyzed the NMR titration data for the Cel9B CBD / cellulose interactions. Interestingly, comparison of best fit with different kinetic models to experimental data suggests that only the state Pa is capable of binding to cellulose. By global fitting to the line-shape data at different ligand concentrations, we have determined the dissociation rate constant koff and the association rate constant kon for the Cel9B CBD / cellulose interactions: koff = 590 s-1 and kon = 2.7 x 107 M-1 s-1 for cellotetraose; and koff = 99 s-1 and kon = 6.7 x 107 M-1 s-1 for cellohexaose. Although association of Cel9B CBD to cellulose is relatively fast for a complex formation that is not driven by electrostatic interactions, dissociation is also fast. The rapid dissociation and association seem suitable for rapid turnover of substrate for the enzymatic reaction.

The kinetic information from our studies provides important insights into the function of the cellulase enzyme Cel9B. The substrate of Cel9B is noncrystalline cellulose, which is amorphous cluster of cellulose chains. When the enzyme attacks saccharide chains in noncrystalline cellulose, the local concentration of possible binding sites around the enzyme is typically as high as 1 mM. Our kinetic data suggest that under such conditions, direct transfer becomes ~4-fold faster than translocation via the “dissociation & re-association” mechanism. Thus, the direct transfer mechanism seems to play a major role in translocation of the enzyme from one substrate site to another. Since the Cel9B CBD is homologous to many other CBDs of other cellulase enzymes, direct transfer is likely a general mechanism that enhances translocation of the enzyme on noncrystalline cellulose. Modulations of this mechanism by engineering may lead to improved activities of the enzymes. 

Currently, we are at the final stage of preparing a manuscript on the kinetic mechanisms for the protein-cellulose interactions. This work is important not only because it provides important insights into the protein-cellulose interactions, but also because it demonstrates for the first time that direct transfer accelerates a biological process other than protein-DNA interactions. We plan to submit the manuscript to PNAS.  In the context of this project, we have developed new NMR methods to study the side-chain dynamics, because the side-chain dynamics should be closely related to the kinetic properties of CBDs. We have published two papers on the method development in Journal of the American Chemical Socieity.

This two-year project involved four members of the PI’s laboratory. A postdoctoral fellow, D. Krishna Rao, and a graduate student, Debashish Sahu, made plasmids for expression of Cel9B CBD and Cel9A CBD, and established protocols for protein purification in our lab. A graduate student, Levani Zandarashivili, prepared isotope-labeled samples of these proteins, and carried out NMR experiments to study the kinetic and dynamic mechanisms for the protein-cellulose interactions. A graduate student, Alexandre Esadze, was involved in development of new NMR methods. Through this project, the participants have considerably improved their scientific knowledge and research skills. This project has also allowed the PI to explore the energy-related research, which was a new direction for him. Successful completion of the project has given him confidence in pursuing more in this field. Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund for support of this research.

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