Polyethylene terephthalate (PET) is one of the most-consumed synthetic polymers, with an annual production of 50 million tons. Unfortunately, PET accumulates as waste and is highly resistant to biodegradation. Recently, fungal and bacterial thermophilic hydrolases were found to catalyze PET hydrolysis with optimal activities at high temperatures. Strikingly, an enzyme from Ideonella sakaiensis, termed PETase, was described to efficiently degrade PET at room temperature, but the molecular basis of its activity is not currently understood. Here, a crystal structure of PETase was determined at 2.02 Å resolution and employed in molecular dynamics simulations showing that the active site of PETase has higher flexibility at room temperature than its thermophilic counterparts. This flexibility is controlled by a novel disulfide bond in its active site, with its removal leading to destabilization of the catalytic triad and reduction of the hydrolase activity. Molecular docking of a model substrate predicts that PET binds to PETase in a unique and energetically favorable conformation facilitated by several residue substitutions within its active site when compared to other enzymes. These computational predictions are in excellent agreement with recent mutagenesis and PET film degradation analyses. Finally, we rationalize the increased catalytic activity of PETase at room temperature through molecular dynamics simulations of enzyme-ligand complexes for PETase and other thermophilic PET-degrading enzymes at 298, 323, and 353 K. Our results reveal that both the binding pose and residue substitutions within PETase favor proximity between the catalytic residues and the labile carbonyl of the substrate at room temperature, suggesting a more favorable hydrolytic reaction. These results are valuable for enabling detailed evolutionary analysis of PET-degrading enzymes and for rational design endeavors aiming at increasing the efficiency of PETase and similar enzymes toward plastic degradation.

Studying chemo-mechanical coupling at interfaces is important for fields ranging from lubrication and tribology to microfluidics and cell biology. Several polymeric macro- and microscopic systems and cantilevers have been developed to image forces at interfaces, but few materials are amenable for molecular tension sensing. To address this issue, we have developed a gold nanoparticle sensor for molecular tension-based fluorescence microscopy (MTFM). As a proof of concept, we imaged the tension exerted by integrin receptors at the interface between living cells and a substrate with high spatial (<1 μm) resolution, at 100 ms acquisition times, and with molecular specificity. We report integrin tension values ranging from 1 to 15 pN and a mean of ~1 pN within focal adhesions. Through the use of a conventional fluorescence microscope, this method demonstrates a force sensitivity that is three orders of magnitude greater than is achievable by traction force microscopy or PDMS micro-post arrays,1 which are the standard in cellular biomechanics.

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ~10(6) - 10(8) times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.

Commensal bacteria that colonize the human colon produce significant amounts of di/tripeptides. We were the first to report that PepT1 transports the small formylated bacterial peptide (fMLP) (1, 2). In the interval since that time, we have shown that other bacterial peptides, such as MDP 3 and Tri-DAP, may also be transported by hPepT1 (3, 4). Small bacterial peptides occur at substantially lower levels in the small intestine compared with the colon in line with the reduced numbers of prokaryotes present in the small intestine of humans. Interestingly, hPepT1 expression is normally restricted to the small intestine, a site in which the levels of small bacterial peptides are low, reflecting the sparse bacterial load of this tissue relative to that of the colon. Thus, the profile of hPepT1 expression along the normal human digestive tract is such that access of small bacterial peptides to hPepT1 minimizes intracellular uptake of these peptides. This normal expression pattern becomes altered in patients with chronic ulcerative colitis or Crohn disease in whom expression of hPepT1 occurs in the colon. The transporter will consequently mediate the intracellular accumulation of small prokaryotic materials. We and others have shown that such accumulation of bacterial products (including

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