The reaction coordinate of a bacterial GH47 α-mannosidase: a combined quantum mechanical and structural approach.

Mannosides in the southern hemisphere: Conformational analysis of enzymatic mannoside hydrolysis informs strategies for enzyme inhibition and inspires solutions to mannoside synthesis. Atomic resolution structures along the reaction coordinate of an inverting α-mannosidase show how the enzyme distorts the substrate and transition state. QM/MM calculations reveal how the free energy landscape of isolated α-D-mannose is molded on enzyme to only allow one conformationally accessible reaction coordinate.

Dendrimers as size selective inhibitors to protein-protein binding.

This communication describes how the "quantized" size effect of dendrimers can be exploited towards a size selective binding mechanism for the inhibition of protein-protein binding.

High-molecular-weight polymers for protein crystallization: poly-gamma-glutamic acid-based precipitants.

Protein crystallization has been revolutionized by the introduction of high-throughput technologies, which have led to a speeding up of the process while simultaneously reducing the amount of protein sample necessary. Nonetheless, the chemistry dimension of protein crystallization has remained relatively undeveloped. Most crystallization screens are based on the same set of precipitants. To address this shortcoming, the development of new protein precipitants based on poly-gamma-glutamic acid (PGA) polymers with different molecular-weight ranges is reported here: PGA-LM (low molecular weight) of approximately 400 kDa and PGA-HM (high molecular weight) of >1,000 kDa. It is also demonstrated that protein precipitants can be expanded further to polymers with much higher molecular weight than those that are currently in use. Furthermore, the modification of PGA-like polymers by covalent attachments of glucosamine substantially improved their solubility without affecting their crystallization properties. Some preliminary PGA-based screens are presented here.

Microscale vapour diffusion for protein crystallization.

The development of new crystallization platforms via the application of high-throughput technologies has delivered a plethora of crystallization plates suitable for robot-driven and manual setups. However, practically all these plates (except for microfluidic channel chips) are based on a very similar design and well (precipitant):drop (protein) volume ratios. A new type of crystallization plate (microplate) has therefore been developed and tested that still employs the classical vapour-diffusion technique but minimizes the precipitant well volume to 1.2 microl for a 150 nl protein drop setup. This enables a very significant saving on the total bulk of the crystallization screen, hence allowing the application of new, rare and expensive solutions in automated crystallization-screening procedures. Additionally, owing to the very low drop:well volume ratio, the new microplate can significantly accelerate the equilibrium time necessary for crystal nucleation and growth, in many cases shortening the high-throughput crystallization screening process to a few hours.

Intramolecular cyclization of diketopiperazine formation in solid-state enalapril maleate studied by thermal FT-IR microscopic system.

The pathway of diketopiperazine (DKP) formation of solid-state enalapril maleate has been studied by using a novel Fourier transform infrared microspectroscope equipped with a thermal analyzer (thermal FT-IR microscopic system). The thermogram of the conventional differential scanning calorimetry (DSC) method was also compared. The results show new evidence of IR peaks at 3250 cm(-1) (the broad O-H stretching mode of water), and at 1738 and 1672 cm(-1) (the carbonyl band of DKP), indicating DKP formation in enalapril maleate via intramolecular cyclization. Moreover, the disappearance of IR peaks from enalapril maleate at 3215 cm(-1) (the secondary amine), 1728 cm(-1) (the carbonyl group of carboxylic acid), and 1649 cm(-1) (the carbonyl stretching of tertiary amide) also confirmed the DKP formation. The thermal FT-IR microscopic system clearly evidenced that the DKP formation in enalapril maleate started from 129 degrees C, and reached a maximum at 137 degrees C. This result was also confirmed by the conventional DSC thermogram of the compressed mixture of KBr powder and enalapril maleate, in which an endothermic peak at 144 degrees C with an extrapolated onset temperature at 137 degrees C was observed. This strongly suggests that the thermal FT-IR microscopic system was able to qualitatively detect the formation of DKP derivatives in solid-state enalapril maleate via intramolecular cyclization.

Hydration-induced proton transfer in the solid state of norfloxacin.

Norfloxacin is a special compound. Its hydrate form seems to be more soluble in water than in the anhydrate form. To investigate the hydration behavior of norfloxacin, the moisture-sorption analysis of anhydrous norfloxacin in different humidities was determined by using differential scanning calorimetry and thermogravimetric analysis. The contents of free water and bound water in the moisture-equilibrated norfloxacin were estimated quantitatively by using a curve-fitting program. Fourier transform infrared microspectroscopy with or without thermal analyzer was used to examine the structural change and dehydration process of norfloxacin in different humidities. The result indicates that the water content sorbed to anhydrous norfloxacin changed lightly below 51% relative humidity (RH) but increased markedly beyond 51% RH. The content of free water in the moisture-equilibrated norfloxacin was nearly to zero below 55% RH, but increased dramatically in high humidity. The content of bound water also enhanced gradually with the external humidity and reached to a constant of one unit after > 75% RH. When norfloxacin anhydrate transformed to its hydrate, the infrared peak intensity at 1732 and 1253 cm(-1) assigned to the C=O and C-O groups of carboxylic acid decreased gradually with the increase of water content, but the infrared peak intensity at 1584 and 1339 cm(-1) corresponding to asymmetric and symmetric carboxylates increased. Furthermore, the peak at 2553 cm(-1) assigned to the NH(+)(2) also appeared clearly and shifted from 2558 cm(-1) in higher water content and humidity. The main functional groups of norfloxacin changed from COOH to COO(-) and NH to NH(+)(2), attributable to the proton transfer from COOH group to NH group. This suggests that the hydration can induce the interaction between norfloxacin molecules from hydrogen bonding to ionic bonding by a proton-transfer process in the solid state.