Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.     

Role of protein in the primary step of the photoreaction of yellow protein

We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment.     

Enzymatic determination of zinc in vegetables using apocarbonic anhydrase

A new enzymatic method has been developed to determine trace amounts of Zn2+ in vegetables. The basis of the method is that apocarbonic anhydrase regains its activity in proportion to the concentration of Zn2+ present in solution. Bovine carbonic anhydrase was purified from erythrocyte haemolysate by affinity chromatography and the bound Zn2+ removed by dialysis of purified enzyme against a solution of pyridine-2, 6-dicarboxylic acid. Pure (100%) apoenzyme was obtained. The concentration of Zn2+ in vegetable samples was determined using the enzymatic method and by atomic absorption spectroscopy. Determinations made using the two methods were not significantly different one from another.     

Glycopolymer charge density determines conformation in human ocular mucin gene products: an atomic force microscope study

Atomic force microscopy (AFM) has been applied to the study of heterogeneity in the structure and function of individual biopolymers with complex structures such as glycoproteins, polysaccharides and nucleic acids. In this work we describe experiments which shed light on the heterogeneity of human ocular mucin gene products. By separating samples of native human ocular mucins on a caesium chloride density gradient, at least three populations consisting predominantly of products of the gene MUC5AC can be identified. Separation on the caesium chloride density gradient is governed by molecular architecture and charge density, and thus provides a route to the discrimination between different glycoforms within a glycoprotein sample. AFM images of these populations show that each is characterised by different conformational properties and polymer diameters, both of which can be attributed to differences in the degree and nature of glycosylation. These differences in glycosylation are likely to be the result of post-translational processing and may also have functional consequences. The AFM's ability to examine the composition of a predominantly single gene product population at the level of the single molecule allows the consequences of post-translational process heterogeneity to be examined at high resolution.     

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.     

A method for testing compressive properties of single proteoglycan aggregates

This study presents a method for direct measurement of the compressive properties of single molecules of proteoglycan aggregate using a state-of-the-art laser tweezers/interferometer system previously developed to test the tensile properties of single molecules. A typical molecule of proteoglycan aggregate showed a highly non-linear resistance to compression after being compressed to about 25% of its original molecule length.     

Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.     

Observing structure,function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5–1 nm and a vertical resolution of 0.1–0.2 nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.     

Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1

Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.