Effects of substrate binding on the dynamics of RNase A: Molecular dynamics simulations of UpA bound and native RNase A

RNase A has been extensively used as a model protein in several biophysical and biochemical studies. Using the available structural and biochemical results, RNase A-UpA interaction has been computationally modeled at an atomic level. In this study, the molecular dynamics (MD) simulations of native and UpA bound RNase A have been carried out. The gross dynamical behavior and atomic fluctuations of the free and UpA bound RNase A have been characterized. Principal component analysis is carried out to identify the important modes of collective motion and to analyze the changes brought out in these modes of RNase A upon UpA binding. The hydrogen bonds are monitored to study the atomic details of RNase A-UpA interactions and RNase A-water interactions. Based on these analysis, the stability of the free and UpA bound RNase A are discussed. © 1997 John Wiley & Sons, Inc. Biopoly 42: 505–520, 1997     

Construction and validation of an atomic model for bacterial TSPO from electron microscopy density,evolutionary constraints,and biochemical and biophysical data

The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications.     

辐射传输模型多尺度反演植被理化参数研究进展

植被是生态系统最重要的组成成分之一,许多与植被有关的物质能量交换过程都与植被的理化参数密切相关,因此定量估算植被的理化参数含量对监测植被生长状况、森林火灾预警以及研究全球碳氮循环过程等都具有重要意义.在众多定量反演植被理化参数的方法中,基于数学、物理学以及生物学的基本理论建立起来的辐射传输模型受到越来越多的关注.辐射传输模型描述了植被与入射辐射之间的相互作用过程和特征,相对于传统的经验/半经验方法,辐射传输模型物理意义明确,具有稳定性和可移植性强的特点.在分析国内外最新相关研究的基础上,首先从植被叶片、冠层和像元3个不同的尺度阐述反演植被理化参数的辐射传输模型.叶片尺度上主要介绍PROSPECT模型和LIBERTY模型;冠层尺度上主要介绍SAIL冠层辐射传输模型以及PROSPECT与SAIL耦合的PROSAIL叶片-冠层辐射传输模型;像元尺度的植被理化参数反演目前主要采用冠层尺度的辐射传输模型.其次,分析尺度变化下植被理化参数遥感反演所面临的主要问题,如不同尺度下模型参数敏感性的变化、辐射传输模型的选取以及混合像元的影响等.最后,总结展望植被理化参数反演多模型与多种数据源相互结合的研究趋势,以及将来具有高空间分辨率的高光谱遥感卫星升空后所带来的发展前景.     

Mathematical aspects of protein structure determination with NMR orientational restraints

The field of structural biology is becoming increasingly important as new technological developments facilitate the collection of data on the atomic structures of proteins and nucleic acids. The solid-state NMR method is a relatively new biophysical technique that holds particular promise for determining the structures of peptides and proteins that are located within the cell membrane. This method provides information on the orientation of the peptide planes relative to an external magnetic field. In this article, we discuss some of the mathematical methods and tools that are useful in deriving the atomic structure from these orientational data. We first discuss how the data are viewed as tensors, and how these tensors can be used to construct an initial atomic model, assuming ideal stereochemistry. We then discuss methods for refining the models using global optimization, with stereochemistry constraints treated as penalty functions. These two processes, initial model building followed by refinement, are the two crucial steps between data collection and the final atomic model.     

Regulation of tumor invasion by interstitial fluid flow

The importance of the tumor microenvironment in cancer progression is undisputed, yet the significance of biophysical forces in the microenvironment remains poorly understood. Interstitial fluid flow is a nearly ubiquitous and physiologically relevant biophysical force that is elevated in tumors because of tumor-associated angiogenesis and lymphangiogenesis, as well as changes in the tumor stroma. Not only does it apply physical forces to cells directly, but interstitial flow also creates gradients of soluble signals in the tumor microenvironment, thus influencing cell behavior and modulating cell-cell interactions. In this paper, we highlight our current understanding of interstitial fluid flow in the context of the tumor, focusing on the physical changes that lead to elevated interstitial flow, how cells sense flow and how they respond to changes in interstitial flow. In particular, we emphasize that interstitial flow can directly promote tumor cell invasion through a mechanism known as autologous chemotaxis, and indirectly support tumor invasion via both biophysical and biochemical cues generated by stromal cells. Thus, interstitial fluid flow demonstrates how important biophysical factors are in cancer, both by modulating cell behavior and coupling biophysical and biochemical signals.     

Feasibility of noninvasive evaluation of biophysical properties of tissue-engineered cartilage by using quantitative MRI

The application of tissue-engineered cartilage in a clinical setting requires a noninvasive method to assess the biophysical and biochemical properties of the engineered cartilage. Since articular cartilage is composed of 70-80% water and has dense extracellular matrixes (ECM), it is considered that the condition of the water molecules in the tissue is correlated with its biomechanical property. Therefore, magnetic resonance imaging (MRI) represents a potential approach to assess the biophysical property of the engineered cartilage. In this study, we test the hypothesis that quantitative MRI can be used as a noninvasive assessment method to assess the biophysical property of the engineered cartilage. To reconstruct a model of cartilaginous tissue, chondrocytes harvested from the humeral head of calves were embedded in an agarose gel and cultured in vitro up to 4 weeks. Equilibrium Young's moduli were determined from the stress relaxation tests. After mechanical testing, MRI-derived parameters (longitudinal relaxation time T1, transverse relaxation time T2, and water self-diffusion coefficient D) were measured. The equilibrium Young's modulus of the engineered cartilage showed a tendency to increase with an increase in the culture time, whereas T1 and D decreased. Based on a regression analysis, T1 and D showed a strong correlation with the equilibrium Young's modulus. The results showed that T1 and D values derived from the MRI measurements could be used to noninvasively monitor the biophysical properties of the engineered cartilage.     

Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to compute the biophysical stimuli generated within a cell under mechanical loading. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of atomic force microscopy indentation was performed and results showed a force/indentation simulation with the range of experimental results. A parametric analysis of both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age) was performed. Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain as compared with young cells, but the difference, surprisingly, is very small and may not be measurable experimentally. Ageing is predicted to have a more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models; correspondence between computed and measured force/displacement behaviours provides a high-level validation of the model. Regarding the effect of ageing, the models suggest only small, although possibly physiologically significant, differences in internal biophysical stimuli between normal and aged cells.     

A convenient and versatile hydroponic cultivation system for Arabidopsis thaliana

A versatile two-step cultivation procedure for Arabidopsis thaliana is described for the production of large quantities of leaf material suitable for biochemical and biophysical analysis. The first step comprises a miniature greenhouse made out of a plastic pipette box to grow the seedlings to the six-leaf stage. For continued growth, the seedlings are transferred to hydroponic cultivation using an opaque container covered by a styrofoam lid. Transfer of the small seedlings to hydroponic culture is facilitated by growth in separate pipette tips, which protects vulnerable roots from damage. The hydroponic cultivation system is easy to scale-up and produces large amounts of relatively large leaves and roots. This hydroponic system produces enough plant material to make Arabidopsis a feasible model for biochemical and biophysical experiments, which can be combined with the available genetic information to address various aspects of plant functional genomics.     

YUP.SCX: coaxing atomic models into medium resolution electron density maps

The structures of large macromolecular complexes in different functional states can be determined by cryo-electron microscopy, which yields electron density maps of low to intermediate resolutions. The maps can be combined with high-resolution atomic structures of components of the complex, to produce a model for the complex that is more accurate than the formal resolution of the map. To this end, methods have been developed to dock atomic models into density maps rigidly or flexibly, and to refine a docked model so as to optimize the fit of the atomic model into the map. We have developed a new refinement method called YUP.SCX. The electron density map is converted into a component of the potential energy function to which terms for stereochemical restraints and volume exclusion are added. The potential energy function is then minimized (using simulated annealing) to yield a stereochemically-restrained atomic structure that fits into the electron density map optimally. We used this procedure to construct an atomic model of the 70S ribosome in the pre-accommodation state. Although some atoms are displaced by as much as 33 Å, they divide themselves into nearly rigid fragments along natural boundaries with smooth transitions between the fragments.     

Investigation of permolecular structure of lipase from Rhizopus niveus

It has been shown by classical biophysical and biochemical methods in combination with atomic microscopy that lipase from Rhizopus niveus exists in a water solution as a dimer with a molecular weight of 96 kDa. The rate of splitting of triglycerides by a dimeric molecules is twice that of monomers. The heat stability of the monomeric form of lipase at temperatures of 20-60 degrees C is significantly higher than that of the native molecule.     

The force-velocity relationship for the actin-based motility of Listeria monocytogenes

The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.     

On contribution of known atomic partial charges of protein backbone in electrostatic potential density maps

Partial charges of atoms in a molecule and electrostatic potential (ESP) density for that molecule are known to bear a strong correlation. In order to generate a set of point‐field force field parameters for molecular dynamics, Kollman and coworkers have extracted atomic partial charges for each of all 20 amino acids using restrained partial charge‐fitting procedures from theoretical ESP density obtained from condensed‐state quantum mechanics. The magnitude of atomic partial charges for neutral peptide backbone they have obtained is similar to that of partial atomic charges for ionized carboxylate side chain atoms. In this study, the effect of these known atomic partial charges on ESP is examined using computer simulations and compared with the experimental ESP density recently obtained for proteins using electron microscopy. It is found that the observed ESP density maps are most consistent with the simulations that include atomic partial charges of protein backbone. Therefore, atomic partial charges are integral part of atomic properties in protein molecules and should be included in model refinement.     

Elucidating the role of matrix stiffness in 3D cell migration and remodeling

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.     

Proteoliposomes in nanobiotechnology

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid–protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid–protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multi-protein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.     

Structure–function relationship in P-type ATPases—a biophysical approach

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure–function relationship.     

Biochemical,biophysical, and genetic changes of porcine trophoblast‐derived stem‐like cells during differentiation as evaluated using Raman microspectroscopy,Atomic force microscopy,and quantitative polymerase chain reaction

Porcine trophoblast‐derived stem‐like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast‐derived cells, cultured in vitro in a defined and non‐serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Young's modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix‐related genes were significantly impacted by medium type (non‐serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy—both considered as label‐free, non‐invasive techniques—can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton‐related genes exhibit similar pattern to that of Young's modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton‐related genes and cellular stiffness. genesis 53:749–761, 2015. © 2015 Wiley Periodicals, Inc.     

Structure and proposed mechanism for the pH-sensing Helicobacter pylori chemoreceptor TlpB

pH sensing is crucial for survival of most organisms, yet the molecular basis of such sensing is poorly understood. Here, we present an atomic resolution structure of the periplasmic portion of the acid-sensing chemoreceptor, TlpB, from the gastric pathogen Helicobacter pylori. The structure reveals a universal signaling fold, a PAS domain, with a molecule of urea bound with high affinity. Through biophysical, biochemical, and in?vivo mutagenesis studies, we show that urea and the urea-binding site residues play critical roles in the ability of H.?pylori to sense acid. Our signaling model predicts that protonation events at Asp114, affected by changes in pH, dictate the stability of TlpB through urea binding.     

Biophysical assessment of single cell cytotoxicity: diesel exhaust particle-treated human aortic endothelial cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs.     

An agent-based model for mRNA export through the nuclear pore complex

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.