Biomedical subjects

Shi-You Ding

Publications and source records attributed to Shi-You Ding.

5 recordsLinked to original sources

PubMed2006

The maize primary cell wall microfibril: a new model derived from direct visualization.

Understanding the molecular architecture of the plant cell wall is critical to reducing the biomass recalcitrance problem, which currently impedes economic bioconversion processing. The parenchyma cell walls from field senesced, maize stem pith have been directly visualized without extraction processes using high-resolution atomic force microscopy (AFM). By imaging the cell wall inner surfaces from different cells and different faces of the same cell, we were able to map the native primary cell wall ultrastructures. Depending on the thickness of non-cellulosic deposition, the parallel-microfibrils appear in various morphologies ranging from clearly defined to completely embedded in the wall matrixes forming cell wall lamella. Macrofibrils were found to exist only on the uppermost layer of the native primary cell wall and appeared to be bundles of elementary fibrils. This novel observation led us to a new hypothesis for the cell wall fibrillar network and biosynthesis processes. Put concisely, a number of elementary fibrils are synthesized at one locus, that of the cellulose synthase complex (CelS), and coalesce into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils with deposition of other cell wall components (i.e. hemicelluloses, pectin, etc.) also evident. On the basis of these AFM surface measurements and current supportive evidence from cell wall biophysics, biosynthesis, and genomics, we propose a new molecular model consisting of a 36-glucan-chain elementary fibril, in which the 36-glucan chains form both crystalline and subcrystalline structures. We also propose a modified model of CelS based on recently reported experimental evidence from plant cell wall biosynthesis.

Cell Wall↗

PubMed2006

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution.

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.

Carbohydrate Metabolism↗

PubMed2003

The bacterial scaffoldin: structure, function and potential applications in the nanosciences.

Natural protein complexes may provide the best templates for nanometer-scale technology and new biomaterials. The bacterial cellulosome is becoming a well-understood multi-protein complex found in cellulolytic microorganisms. The scaffoldin subunits of the bacterial cellulosome function to organize and position other protein subunits into the complex. The scaffoldins can also serve as an attachment device for harnessing the cellulosome onto the cell surface and/or for its targeting to substrate. Biochemical and molecular biological evidence have identified a receptor/adaptor type of protein domain pair, called "cohesin and dockerin," which is responsible for cellulosome self-assembly. The recognition between cohesin and dockerin is generally type and/or species specific. More than 80 cohesin and 100 dockerin sequences have been found, mostly from anaerobic bacteria. X-ray crystallography and NMR have been used to determine the three-dimensional structures of representative cohesin and dockerin domains, respectively. The cohesin peptide is about 140 amino acids in length and highly conserved in sequence and domain structure. The dockerin domain comprises about 70 amino acids and contains two 22 amino acid duplicated regions, each of which includes an "F-hand" modification of the EF-hand calcium-binding motif. Biochemical evidence and site-directed mutagenesis have confirmed that the two F-hand motifs are required for function and calcium dependence; at least two amino acids from each motif are critical for cohesin-dockerin recognition. In this report, we review the structure and function of the scaffoldin of the bacterial cellulosome and emphasize a detailed sequence analysis of the cohesin and dockerin domains. We also speculate about potential applications in nanoscience that may be based on cohesin-dockerin recognition.

Bacteria↗

PubMed2003

The cellulosome system of Acetivibrio cellulolyticus includes a novel type of adaptor protein and a cell surface anchoring protein.

A scaffoldin gene cluster was identified in the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus. The previously described scaffoldin gene, cipV, encodes an N-terminal family 9 glycoside hydrolase, a family 3b cellulose-binding domain, seven cohesin domains, and a C-terminal dockerin. The gene immediately downstream of cipV was sequenced and designated scaB. The protein encoded by this gene has 942 amino acid residues and a calculated molecular weight of 100,358 and includes an N-terminal signal peptide, four type II cohesions, and a C-terminal dockerin. ScaB cohesins 1 and 2 are very closely linked. Similar, but not identical, 39-residue Thr-rich linker segments separate cohesin 2 from cohesin 3 and cohesin 3 from cohesin 4, and an 84-residue Thr-rich linker connects the fourth cohesin to a C-terminal dockerin. The scaC gene downstream of scaB codes for a 1,237-residue polypeptide that includes a signal peptide, three cohesins, and a C-terminal S-layer homology (SLH) module. A long, ca. 550-residue linker separates the third cohesin and the SLH module of ScaC and is characterized by an 18-residue Pro-Thr-Ala-Ser-rich segment that is repeated 27 times. The calculated molecular weight of the mature ScaC polypeptide (excluding the signal peptide) is 124,162. The presence of the cohesins and the conserved SLH module implies that ScaC acts as an anchoring protein. The ScaC cohesins are on a separate branch of the phylogenetic tree that is close to, but distinct from, the type I cohesins. Affinity blotting with representative recombinant probes revealed the following specific intermodular interactions: (i) an expressed CipV cohesin binds selectively to an enzyme-borne dockerin, (ii) a representative ScaB cohesin binds to the CipV band of the cell-free supernatant fraction, and (iii) a ScaC cohesin binds to the ScaB dockerin. The experimental evidence thus indicates that CipV acts as a primary (enzyme-recognizing) scaffoldin, and the protein was also designated ScaA. In addition, ScaB is thought to assume the role of an adaptor protein, which connects the primary scaffoldin (ScaA) to the cohesin-containing anchoring scaffoldin (ScaC). The cellulosome system of A. cellulolyticus thus appears to exhibit a special type of organization that reflects the function of the ScaB adaptor protein. The intercalation of three multiple cohesin-containing scaffoldins results in marked amplification of the number of enzyme subunits per cellulosome unit. At least 96 enzymes can apparently be incorporated into an individual A. cellulolyticus cellulosome. The role of such amplified enzyme incorporation and the resultant proximity of the enzymes within the cellulosome complex presumably contribute to the enhanced synergistic action and overall efficient digestion of recalcitrant forms of cellulose. Comparison of the emerging organization of the A. cellulolyticus cellulosome with the organizations in other cellulolytic bacteria revealed the diversity of the supramolecular architecture.

Amino Acid Sequence↗

PubMed2003

Novel organization and divergent dockerin specificities in the cellulosome system of Ruminococcus flavefaciens.

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an approximately 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Animals↗