Transient partial unfolding in Escherichia coli dihydrofolate reductase investigated by native state proteolysis

Joseph Robert Kasper, Purdue University

Abstract

A protein's amino acid sequence encodes its conformational energy landscape. The energy landscape is shaped by the probability of each conformation that the chain can occupy and directs the folding of the chain. The folded protein is highly populated at equilibrium under native conditions; however, transient populations of non-native partially or globally unfolded conformations also exist in equilibrium with the folded protein. Detecting the population of transient, high-energy forms requires a method selective for those forms. We have used native-state proteolysis, detecting the lowest energy non-native form that can be cleaved by a nonspecific protease. Using native-state proteolysis, we uncovered a cleavable form of Escherichia coli dihydrofolate reductase (DHFR) and mapped the unfolded region of the cleavable form. This partially unfolded form of DHFR has free energy, solvent exposure, and structural characteristics that resemble a folding intermediate. A protein's chemical environment, including the presence of ligands, can reshape the protein's energy landscape. After revealing a partially unfolded form of DHFR, we investigated the effect of NADP+ on the energy landscape of DHFR. Again we employed native-state proteolysis, monitoring the affect of NADP+ on proteolysis of DHFR. We found that the cleavable form binds NADP+ with a distinct, lower affinity than the affinity with which the native form binds NADP+. As a result, NADP+ stabilizes native DHFR relative to cleavable DHFR while the cleavable form is still stabilized relative to globally unfolded DHFR. We have used the nonspecific proteases, thermolysin and subtilisin, to perform native-state proteolysis. We discovered that the apparent first-order rate constant for proteolysis increases as the concentration of DHFR decreases. Such an increase in rate constant was highly unexpected because unfolding of DHFR is unimolecular; therefore the concentration of DHFR should not affect the proteolysis rate constant. We followed our observation by investigating the effects of substrate concentration and of added cleavage products on the reaction of these proteases with the protein substrates of E. coli dihydrofolate reductase and ribonuclease H. The species responsible for the inhibition we observed are the cleavage products formed from cleavage of intact proteins. A competitive inhibition model can accurately describe our observations.

Degree

Ph.D.

Advisors

Park, Purdue University.

Subject Area

Molecular biology|Biochemistry|Biophysics

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