Metal-Organic Materials (MOMs) Enhance Proteolytic Selectivity, Efficiency, and Reusability of Trypsin: A Time-Resolved Study on Proteolysis.

Proteases are involved in essential biological functions in nature and have become drug targets recently. In spite of the promising progress, two challenges, (i) the intrinsic instability and (ii) the difficulty in monitoring the catalytic process in real time, still hinder the further understanding and engineering of protease functionalities. These challenges are caused by the lack of proper materials/approaches to stabilize proteases and monitor proteolytic products (truncated polypeptides) in real time in a highly heterogeneous reaction mixture. This work combines metal-organic materials (MOMs), site-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy, and mass spectrometry (MS) to overcome both barriers. A model protease, trypsin, which cleaves the peptide bonds at lysine or arginine residues, was immobilized on a Ca-MOM via aqueous-phase, one-pot cocrystallization, which allows for trypsin protection and ease of separation from its proteolytic products. Time-resolved EPR and MS were employed to monitor the populations, rotational motion, and sequences of the cleaved peptide truncations of a model protein substrate as the reaction proceeded. Our data suggest a significant (at least 5-10 times) enhancement in the catalytic efficiency (kcat/km) of trypsin@Ca-MOM and excellent reusability as compared to free trypsin in solution. Surprisingly, entrapping trypsin in Ca-MOMs results in cleavage site/region selectivity against the protein substrate, as compared to the near nonselective cleavage of all lysine and arginine residues of the substrate in solution. Remarkably, immobilizing trypsin allows for the separation and, thus, MS study on the sequences of truncated peptides in real time, leading to a time-resolved "movie" of trypsin proteolysis. This work demonstrates the use of MOMs and cocrystallization to enhance the selectivity, catalytic efficiency, and stability of trypsin, suggesting the possibility of tuning the catalytic performance of a general protease using MOMs.

Molecular interaction mechanism and structure-activity relationships of protein-polyphenol complexes revealed by side-directed spin labeling-electron paramagnetic resonance (SDSL-EPR) spectroscopy.

Deciphering interactions between bioactive protein and polyphenols are critical for designing and controlling functional protein-polyphenol complexes. Herein, using the site-directed spin labeled T4 lysozyme (T4L) and rosmarinic acid (RA) as a model system, we combined electron paramagnetic resonance spectra to investigate molecular interaction mechanism of the protein-polyphenol complexes in structural or conformational details. Experimental results show that molecular interactions between T4L and RA are a process from order to disorder. TEM images display that the complexes finally assemble into quasi-spherical colloidal particles. When T4L/RA ratio is 1:1, the complexes exhibit the optimized enzymatic and antioxidant dual-functionalities due to the synergetic effect and protection mechanism. However, with excess addition of RA, the enzymatic and antioxidant activities of the complexes started to attenuate because the catalytic active site and bioactive hydroxyl groups were buried. The revealed high-resolution interaction process could help better understand the corresponding alterations between structure and functionalities.

Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.

Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2 µM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein-protein interactions with minor adaptations.

A Combined Spectroscopic and In Silico Approach to Evaluate the Interaction of Human Frataxin with Mitochondrial Superoxide Dismutase.

Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich's ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron-sulfur cluster machinery, redox imbalance, and iron homeostasis. FXN is synthesized in the cytosol and imported into the mitochondria, where it is proteolytically cleaved to the mature form. Its involvement in the redox imbalance suggests that FXN could interact with mitochondrial superoxide dismutase (SOD2), a key enzyme in antioxidant cellular defense. In this work, we use site-directed spin labelling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) and fluorescence quenching experiments to investigate the interaction between human FXN and SOD2 in vitro. Spectroscopic data are combined with rigid body protein-protein docking to assess the potential structure of the FXN-SOD2 complex, which leaves the metal binding region of FXN accessible to the solvent. We provide evidence that human FXN interacts with human SOD2 in vitro and that the complex is in fast exchange. This interaction could be relevant during the assembly of iron-sulfur (FeS) clusters and/or their incorporation in proteins when FeS clusters are potentially susceptible to attacks by reactive oxygen species.

Influenza A M2 protein conformation depends on choice of model membrane.

While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein. Here we show using site-directed spin-label EPR spectroscopy (SDSL-EPR) that the conformations of M2 peptides in membrane bilayers are clearly influenced by the lipid composition of the bilayers. Altering the bilayer thickness or the lateral pressure profile within the bilayer membrane changes the M2 conformation observed. The multiple M2 peptide conformations observed here, and in other published studies, optimistically may be considered conformations that are sampled by the protein at various stages during influenza infectivity. However, care should be taken that the heterogeneity observed in published structures is not simply an artifact of the choice of the model membrane.