Exploration of CP12 conformational changes and of quaternary structural properties using electrospray ionization traveling wave ion mobility mass spectrometry.

RATIONALE: CP12 is a small chloroplast protein involved in the Benson-Calvin cycle. Since it was demonstrated that the CP12 protein shared different conformational properties between reduced and oxidized states we took advantage of the segregational properties of the Traveling Wave Ion Mobility (TWIM) guide to study subtle conformational changes related to redox changes. METHODS: Electrospray ionization mass (ESI-MS) spectra of the CP12 protein were recorded in the positive ion mode using an ESI source fitted on a quadrupole time-of-flight (QToF) hybrid mass spectrometer equipped with a TWIM cell (Synapt HDMS G1, Waters Corp., Manchester) under non-denaturing conditions. Non-covalent experiments were performed using the same instrument without the use of the TWIM device. RESULTS: Whatever the CP12 form studied, our results showed that CP12 protein was represented by two conformers in equilibrium that displayed very slight differences. These observations led us to propose that CP12 protein structure is rather undergoing transient subtle structural changes than having two different conformational populations in solution. In addition, using non-denaturing experiments, NAD and CP12 stoichiometry were determined with respect to the GAPDH tetramer and the redox state of CP12. CONCLUSIONS: In this study we showed that the use of the segregational property of the ion mobility (TWIM, Synapt G1 HDMS, Waters, Manchester, UK) allowed differentiation of subtle conformational changes between redox states of the CP12 protein. Standard non-denaturing experiments revealed different binding stoichiometry according to the redox state of the CP12 protein.

Molecular mechanism of NADPH-glyceraldehyde-3-phosphate dehydrogenase regulation through the C-terminus of CP12 in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) consists of four GapA subunits. This A4 GAPDH is not autonomously regulated, as the regulatory cysteine residues present on GapB subunits are missing in GapA subunits. The regulation of A4 GAPDH is provided by another protein, CP12. To determine the molecular mechanisms of regulation of A4 GAPDH, we mutated three residues (R82, R190, and S195) of GAPDH of C. reinhardtii. Kinetic studies of GAPDH mutants showed the importance of residue R82 in the specificity of GAPDH for NADPH, as previously shown for the spinach enzyme. The cofactor NADPH was not stabilized through the 2'-phosphate by the serine 195 residue of the algal GAPDH, unlike the case in spinach. The mutation of R190 also led to a structural change that was not observed in the spinach enzyme. This mutation led to a loss of activity for NADPH and NADH, indicating the crucial role of this residue in maintaining the algal GAPDH structure. Finally, the interaction between GAPDH mutants and wild-type and mutated CP12 was analyzed by immunoblotting experiments, surface plasmon resonance, and kinetic studies. The results obtained with these approaches highlight the involvement of the last residue of CP12, Asp80, in modulating the activity of GAPDH by preventing access of the cofactor NADPH to the active site. These results help us to bridge the gap between our knowledge of structure and our understanding of functional biology in GAPDH regulation.

Amplitude of pancreatic lipase lid opening in solution and identification of spin label conformational subensembles by combining continuous wave and pulsed EPR spectroscopy and molecular dynamics.

The opening of the lid that controls the access to the active site of human pancreatic lipase (HPL) was measured from the magnetic interaction between two spin labels grafted on this enzyme. One spin label was introduced at a rigid position in HPL where an accessible cysteine residue (C181) naturally occurs. A second spin label was covalently bound to the mobile lid after introducing a cysteine residue at position 249 by site-directed mutagenesis. Double electron-electron resonance (DEER) experiments allowed the estimation of a distance of 19 +/- 2 A between the spin labels when bilabeled HPL was alone in a frozen solution, i.e., with the lid in the closed conformation. A magnetic interaction was however detected by continuous wave EPR experiments, suggesting that a fraction of bilabeled HPL contained spin labels separated by a shorter distance. These results could be interpreted by the presence of two conformational subensembles for the spin label lateral chain at position 249 when the lid was closed. The existence of these conformational subensembles was revealed by molecular dynamics experiments and confirmed by the simulation of the EPR spectrum. When the lid opening was induced by the addition of bile salts and colipase, a larger distance of 43 +/- 2 A between the two spin labels was estimated from DEER experiments. The distances measured between the spin labels grafted at positions 181 and 249 were in good agreement with those estimated from the known X-ray structures of HPL in the closed and open conformations, but for the first time, the amplitude of the lid opening was measured in solution or in a frozen solution in the presence of amphiphiles.

Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy.

The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.

Assessing induced folding of an intrinsically disordered protein by site-directed spin-labeling electron paramagnetic resonance spectroscopy.

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.

CP12 residues involved in the formation and regulation of the glyceraldehyde-3-phosphate dehydrogenase-CP12-phosphoribulokinase complex in Chlamydomonas reinhardtii.

CP12, a member of the intrinsically disordered protein family, forms a stable complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). To understand the function of conserved residues of CP12 in the formation of the GAPDH-CP12-PRK complex and in the regulation of the enzymes within this complex, we have produced mutants of CP12 by site-directed mutagenesis. The GAPDH, CP12 and PRK recombinant proteins are able to reconstitute spontaneously the ternary complex that has been described in Chlamydomonas reinhardtii. Our analysis reveals that the central part ((35)WXXVEE(47)) of CP12 is required to form the GAPDH-CP12-PRK complex. Using the same series of single amino acid replacements, we have identified individual residues, which seem to represent also contact points for GAPDH. Most notably, substitution of glutamate 74 prevents the binding of GAPDH to CP12. This is similar to the mutant C66S, with which the GAPDH-CP12-PRK complex is not formed. In contrast, replacement of the three last residues ((78)YED(80)) of CP12 has no effect on the formation of the ternary supra-molecular complex. However, our findings strongly suggest that Y78 and D80 are involved in the regulation of the GAPDH activity within the supra-molecular complex, since the mutants, D80K and Y78S, do not down-regulate the activity of GAPDH. The replacement of the amino acid E79 weakens the interaction between GAPDH and CP12 as no GAPDH-CP12 sub-complex is formed. In this case, nevertheless, the supra-molecular complex is formed when PRK is present indicating that PRK strengthens the interaction between GAPDH and CP12 within the supra-molecular complex.

Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy.

Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.