Nanomechanical Properties of Tenascin-X Revealed by Single-Molecule Force Spectroscopy

Tenascin-X is an extracellular matrix protein and binds a variety of molecules in extracellular matrix and on cell membrane. Tenascin-X plays important roles in regulating the structure and mechanical properties of connective tissues. Using single-molecule atomic force microscopy, we have investigated the mechanical properties of bovine tenascin-X in detail. Our results indicated that tenascin-X is an elastic protein and the fibronectin type III (FnIII) domains can unfold under a stretching force and refold to regain their mechanical stability upon the removal of the stretching force. All the 30 FnIII domains of tenascin-X show similar mechanical stability, mechanical unfolding kinetics, and contour length increment upon domain unfolding, despite their large sequence diversity. In contrast to the homogeneity in their mechanical unfolding behaviors, FnIII domains fold at different rates. Using the 10th FnIII domain of tenascin-X (TNXfn10) as a model system, we constructed a polyprotein chimera composed of alternating TNXfn10 and GB1 domains and used atomic force microscopy to confirm that the mechanical properties of TNXfn10 are consistent with those of the FnIII domains of tenascin-X. These results lay the foundation to further study the mechanical properties of individual FnIII domains and establish the relationship between point mutations and mechanical phenotypic effect on tenascin-X. Moreover, our results provided the opportunity to compare the mechanical properties and design of different forms of tenascins. The comparison between tenascin-X and tenascin-C revealed interesting common as well as distinguishing features for mechanical unfolding and folding of tenascin-C and tenascin-X and will open up new avenues to investigate the mechanical functions and architectural design of different forms of tenascins.     

HCV Antibody Response and Genotype Distribution in Different Areas and Races of China

Hepatitis C virus (HCV) heterogeneity accounts for the failure of effective vaccine development and the lack of successful anti-viral therapy in some patients. Little is known about the immune response to HCV peptides and the region or race specific genotypes in China. The objective of this study was to characterize HCV antibody immune response to HCV peptides and HCV genotypes in different regions and races of China. A total of 363 serum samples were collected from HCV carriers in 6 regions in China. The immune response to HCV peptides was evaluated by ELISA. HCV genotypes were examined using nested RT-PCR. We found that the anti-HCV antibody neutralization rates were significantly different among the serum samples from different areas or from different races in the same area. For samples from Tibet and Sinkiang, the rates of neutralization by HCV peptides were only 3.2% and 30.8%, respectively. The genotypes of samples from Tibet and Sinkiang were apparently heterogeneic and included type I, II, III and multiple types (I/II/III, I/II, I/III, II/III). One specific sample with multiple-genotype (I/II/III) HCV infection was found to consist of type I, II, III, II/III and an unclassified genotype. These studies indicate that the anti-HCV antibody immune response to HCV peptides varied across regions and among races. The distribution of HCV genotypes among Tibetans in Tibet and Uighurs in Sinkiang was different from that in the inner areas of China. In addition, a “master” genotype, type II, was found to exist in HCV infection with multiple HCV genotypes.     

An Exploration of the Relations between External Representations and Working Memory

It is commonly hypothesized that external representations serve as memory aids and improve task performance by means of expanding the limited capacity of working memory. However, very few studies have directly examined this memory aid hypothesis. By systematically manipulating how information is available externally versus internally in a sequential number comparison task, three experiments were designed to investigate the relation between external representations and working memory. The experimental results show that when the task requires information from both external representations and working memory, it is the interaction of information from the two sources that determines task performance. In particular, when information from the two sources does not match well, external representations hinder instead of enhance task performance. The study highlights the important role the coordination among different representations plays in distributed cognition. The general relations between external representations and working memory are discussed.     

Grazing and growth responses of a marine oligotrichous ciliate fed with two nanoplankton: does food quality matter for micrograzers?

To investigate whether predator growth and grazing would depend on prey properties besides size, we studied the numerical and functional responses of a marine oligotrichous ciliate isolated in Hong Kong coastal waters, Strobilidium sp., on two nanoplanktonic preys of similar size. The growth and ingestion rates of Strobilidium sp. could be fit with prey concentrations by hyperbolic curves. Strobilidium sp. exhibited higher maximal growth rates and gross growth efficiencies, and lower maximal clearance rates on Nannochloropsis sp. than on Isochrysis galbana. Our results demonstrate that prey properties presumably food quality can have a considerable effect on predator growth and grazing and implications on phytoplankton community structure and biogeochemical cycling.     

Pentacyclic triterpenes. Part 3: Synthesis and biological evaluation of oleanolic acid derivatives as novel inhibitors of glycogen phosphorylase

Oleanolic acid and its synthetic derivatives have been identified as novel inhibitors of glycogen phosphorylase. Within this series of compounds, 4 (IC50 = 3.3 microM) is the most potent GPa inhibitor. Preliminary structure-activity relationships of the oleanolic acid derivatives are discussed.     

Pentacyclic triterpenes. Part 2: Synthesis and biological evaluation of maslinic acid derivatives as glycogen phosphorylase inhibitors

The synthesis of a series of maslinic acid derivatives is described and their effect on rabbit muscle glycogen phosphorylase a evaluated. Within this series of compounds, 15 (IC(50)=7 microM) is the most potent GPa inhibitor. SAR of the maslinic acid derivatives are discussed.     

Improved 3D-QSAR CoMFA of the dopamine transporter blockers with multiple conformations using the genetic algorithm

A 3D-QSAR/CoMFA was performed for a series of 42 piperidine-based dopamine transporter (DAT) blockers. The overall process consisted of three major steps: (1) a pharmacophore model was built using the Genetic Algorithm Similarity Program (GASP); (2) the Flexible Superposition (FlexS) technique was applied to generate multiple conformations for each of the ligands based on the pharmacophore; (3) the Genetic Algorithm was employed to optimize the selection of the ligand conformations for the CoMFA modeling. The CoMFA models were found to be more detailed in the putative binding site by exploring multiple conformations of each ligand. The comparison of the contour maps shows that, in general, these models are comparable and the differences between them result from the ability of the flexible 3-substituents of the ligands to adopt multiple conformations satisfying the same pharmacophore model. These findings provide guidance for the design and improvement of compounds with DAT activity, which is important for the development of a treatment of cocaine addiction and certain neurological disorders.     

Single molecule force spectroscopy reveals a weakly populated microstate of the FnIII domains of tenascin

The native states of proteins exist as an ensemble of conformationally similar microstates. The fluctuations among different microstates are of great importance for the functions and structural stability of proteins. Here, we demonstrate that single molecule atomic force microscopy (AFM) can be used to directly probe the existence of multiple folded microstates. We used the AFM to repeatedly stretch and relax a recombinant tenascin fragment TNfnALL to allow the fibronectin type III (FnIII) domains to undergo repeated unfolding/refolding cycles. In addition to the native state, we discovered that some FnIII domains can refold from the unfolded state into a previously unrecognized microstate, N* state. This novel state is conformationally similar to the native state, but mechanically less stable. The native state unfolds at approximately 120 pN, while the N* state unfolds at approximately 50 pN. These two distinct populations of microstates constitute the ensemble of the folded states for some FnIII domains. An unfolded FnIII domain can fold into either one of the two microstates via two distinct folding routes. These results reveal the dynamic and heterogeneous picture of the folded ensemble for some FnIII domains of tenascin, which may carry important implications for the mechanical functions of tenascins in vivo.     

Dynamics of protein folding and cofactor binding monitored by single-molecule force spectroscopy

Many proteins in living cells require cofactors to carry out their biological functions. To reach their functional states, these proteins need to fold into their unique three-dimensional structures in the presence of their cofactors. Two processes, folding of the protein and binding of cofactors, intermingle with each other, making the direct elucidation of the folding mechanism of proteins in the presence of cofactors challenging. Here we use single-molecule atomic force microscopy to directly monitor the folding and cofactor binding dynamics of an engineered metal-binding protein G6-53 at the single-molecule level. Using the mechanical stability of different conformers of G6-53 as sensitive probes, we directly identified different G6-53 conformers (unfolded, apo- and Ni²⁺-bound) populated along the folding pathway of G6-53 in the presence of its cofactor Ni²⁺. By carrying out single-molecule atomic force microscopy refolding experiments, we monitored kinetic evolution processes of these different conformers. Our results suggested that the majority of G6-53 folds through a binding-after-folding mechanism, whereas a small fraction follows a binding-before-folding pathway. Our study opens an avenue to utilizing force spectroscopy techniques to probe the folding dynamics of proteins in the presence of cofactors at the single-molecule level, and we anticipated that this method can be used to study a wide variety of proteins requiring cofactors for their function.