Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. Here, we describe a general method to accelerate enzymes using pressure waves contained within thin films. Each enzyme responds best to specific frequencies of pressure waves, and we report acceleration landscapes for each protein. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat) and enzymatic efficiency (kcat/Km). Four enzymes displayed an average seven-fold acceleration with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement through this approach. In solving a common problem in enzyme catalysis, we have uncovered a powerful, generalizable tool for enzyme acceleration. This research provides new insights into previously uncontrolled factors affecting enzyme function.

Enzymes catalyze chemical transformations with outstanding stereo-and regio-specificities,b ut many enzymes are limited by their long reaction times.Ageneral method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves,a nd an acceleration landscape for each protein is reported. Avortex fluidic device introduces pressure waves that drive increased rate constants (k cat )a nd enzymatic efficiency (k cat /K m ). Four enzymes displayed an average seven-fold acceleration, with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement using this approach. In solving acommon problem in enzyme catalysis,apowerful, generalizable tool for enzyme acceleration has been uncovered. This researchprovides new insights into previously uncontrolled factors affecting enzyme function.Enzymes make life possible by catalyzing diverse and challenging chemical transformations with exquisite specificity.Applications in both industry [1] and academia [2] rely on the selectivity and power of enzymes to catalyze otherwise challenging transformations.B iocatalysts offer remarkable rate accelerations compared to the uncatalyzed reactions, with typical rate accelerations (k cat /k uncat )o f1 0 5 -t o1 0 15 -fold faster. [3] Though some enzymes are diffusion-limited, [4] the catalytic rates of enzymes are more typically limited by their catalytic efficiency( k cat /K m ); additionally,m olecular crowding, along with product and substrate inhibition, can reduce enzyme efficiency. [5] Though some enzymes catalyse transformations with rapid rates (for example,laccases,fumarases, and alcohol dehydrogenases), [6] other enzymes operate at only modest reaction rates,requiring long reaction times and carefully optimized conditions;for example,DERA requires long processing times (hours to days), and is substrateinhibited. [7] We report aprocess that accelerates four different enzymes at standard temperature and pressure,b ut many other water-soluble enzymes could be accelerated as well.Recently,vortex fluidic devices (VFDs) have been used to accelerate covalent and noncovalent bond formation. VFDs process solutions in thin films by the rapid rotation of as ample tube (Figure 1). [8] Within the thin film, species are subjected to high levels of shear stress,m ass transfer,a nd vibrational energy input at specific rotational speeds.F or example,the VFD demonstrated the effective folding of four different proteins within minutes at standard temperature and pressure. [9] TheV FD has also been used to improve the synthesis of lidocaine [10] and several other organic transformations. [11] In acontinuous flow regime,flow rates of up to 20 mL min À1 can be achieved to process up to 30 Lper day in the current benchtop configuration. Since VFD processing increased the rates of organic reactions and protein folding, we hypothesized that biocatalysis,w hich requires both reactivity and the correct protein fold, could benefit as well.Control r...

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein’s initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

This study examines from a numerical point of view the phenomenon of collisions between the walls of hexcans caused by an explosion in the central hexcan. The scope is both to check the actual resistance of the assembly and to demonstrate the reliability of the calculation method for the effects of collisions between walls.

Solid-state NMR and EPR spectroscopy are important biophysical techniques that are being using to study membrane proteins. CW and pulsed EPR spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. Strategically placed spin-labels alter relaxation times of NMR active nuclei and yield pertinent structural information. EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) are powerful structural biology tools. The DEER technique can be used to measure distances between 2 spin labels from 20 to 70 Å . However, the application of DEER spectroscopy to study membrane proteins can be difficult due to short phase memory times (T m ) and weak DEER modulation in more biologically relevant proteoliposomes when compared to water soluble proteins or membrane proteins in detergent micelles. The combination of these factors often leads to broad distance distributions, poor signal to noise, and limitations in the determination of longer distances. The short phase memory times are typically due to uneven distributions of spin-labeled protein within the lipid bilayer, which creates local inhomogeneous pockets of high spin concentrations. Approaches to overcome these limitations and improve the quality of DEER measurements for membrane proteins will be discussed: lipodisq nanoparticles, bifunctional spin labels (BSL), and Q-band pulsed EPR spectroscopy. ESEEM data will be used to probe the secondary structure of membrane proteins. ESEEM can be thought of as EPR detected NMR. CW-EPR spectra of spin-labeled membrane proteins will be used to investigate dynamics and the immersion depth in a lipid bilayer. A variety of different membrane proteins will be probed with these state-of-the art magnetic resonance techniques. 282-PosStructural and Functional Role of the Surface-Exposed Loops of Ail During Complement-Mediated Evasion by Y. pestis