Nanobiomechanics of proteins and biomembrane

A review of the work done in the Laboratory of Biodynamics of Tokyo Institute of Technology in the last decade has been summarized in this article in relation to the results reported from other laboratories. The emphasis here is the application of nanomechanics based on the force mode of atomic force microscopy (AFM) to proteins and protein-based biological structures. Globular proteins were stretched in various ways to detect the localized rigidity inside of the molecule. When studied by this method, bovine carbonic anhydrase II (BCA II), calmodulin and OspA protein all showed the presence of localized rigid structures inside the molecules. Protein compression experiments were done on BCA II to obtain an estimate of the Young modulus and its change in the process of denaturation. Then, the AFM probe method was turned on to cell membranes and cytoplasmic components. Force curves accompanying the extraction process of membrane proteins from intact cells were analysed in relation to their interaction with the cytoskeletal components. By pushing the AFM probe further into the cytoplasm, mRNAs were recovered from a live cell with minimal damage, and multiplied using PCR technology for their identification. Altogether, the work introduced here forms the basis of nanomechanics of protein and protein-based biostructures and application of the nanomechanical technology to cell biology.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.     

Origin of mechanical strength of bovine carbonic anhydrase studied by molecular dynamics simulation

The forced unfolding process of bovine carbonic anhydrase II (BCA II) was examined at the atomic level by the molecular dynamics (MD) simulation. By force spectroscopy, experimentally obtained force-extension curves (F-E curves) showed a prominent force peak after 50 nm extension. F-E curves obtained from our simulation had three force peaks appearing after extensions of 10-17 nm, 40 nm, and 53 nm, each signifying a brittle fracture of a specific local structure. Upon undergoing the final fracture at 53 nm of extension, the entire molecule became a single flexible chain and was further extended to its full theoretical length, almost as a random coil. This feature of the 53-nm peak strongly suggested its close correspondence to the experimentally observed force peak at approximately 60-nm extension. The 53-nm peak in the molecular dynamics simulation corresponded to the unfolding process of the beta-sheeted core that includes zinc-coordinating histidine residues. These results suggest that the structural change occurring at 50-60 nm in atomic force microscopy experiments corresponded to the destruction of the zinc coordination site.     

Effect of indenter size on elastic modulus of cartilage measured by indentation

Our preliminary indentation experiments showed that the equilibrium elastic modulus of murine tibial cartilage increased with decreasing indenter size: flat-ended 60 deg conical tips with end diameters of 15 microm and 90 microm gave 1.50+/-0.82 MPa (mean+/-standard deviation) and 0.55+/-0.11 MPa, respectively (p<0.01). The goal of this paper is to determine if the dependence on tip size is an inherent feature of the equilibrium elastic modulus of cartilage as measured by indentation. Since modulus values from nonindentation tests are not available for comparison for murine cartilage, bovine cartilage was used. Flat-ended conical or cylindrical tips with end diameters ranging from 5 microm to 4 mm were used to measure the equilibrium elastic modulus of bovine patellar cartilage. The same tips were used to test urethane rubber for comparison. The equilibrium modulus of the bovine patellar cartilage increased monotonically with decreasing tip size. The modulus obtained from the 2 mm and 4 mm tips (0.63+/-0.21 MPa) agreed with values reported in the literature; however, the modulus measured by the 90 microm tip was over two and a half times larger than the value obtained from the 1000 microm tip. In contrast, the elastic modulus of urethane rubber obtained using the same 5 microm-4 mm tips was independent of tip size. The equilibrium elastic modulus of bovine patellar cartilage measured by indentation depends on tip size. This appears to be an inherent feature of indentation of cartilage, perhaps due to its inhomogeneous structure.     

Force and flexibility of flailing myxobacteria

Myxococcus xanthus is a common Gram-negative bacterium that moves by a process called gliding motility. In myxobacteria, two distinct mechanisms for gliding have been discovered. S-type motility requires the extension, attachment, and retraction of type IV pili. The other mechanism, designated as A-type motility, may be driven by the secretion and swelling of slime; however, experiments to confirm or refute this model are still lacking and the force exerted by this mechanism has not been measured. A previously published experiment found that when an M. xanthus cell became stuck at one end, the cell underwent flailing motions. Based on this experiment, I propose an elastic model that can estimate the force produced by the A-motility engine and the bending modulus of a single myxobacterial cell. The model estimates a bending modulus of 3 x 10(-14) erg cm and a force between 50-150 pN. This force is comparable to that predicted by slime extrusion, and the bending modulus is 30-fold smaller than that measured in Bacillus subtilis. This model suggests experiments that can further quantify this process.     

Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.

Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Identification of novel fibronectin fragments detected specifically in juvenile urine

Ugl-Y (young age-related urinary glycoprotein) is an age-specific protein that we have previously identified in urine from healthy subjects under 18 years of age. Isoelectric focusing analysis of Ugl-Y gives a set of three bands, Y1, Y2 and Y3, in the pH region around 3. To determine the complete structure of Ugl-Y, purified Y1 and Y2 from pediatric urine were enzymatically cleaved, and the resulting peptides were analyzed by protein sequencing and/or MALDI-TOF MS. As a result, it was demonstrated that Y1 consists of 189 amino acid residues, and is identical to the region from A723 to R911 of fibronectin, whereas Y2 consists of 181 amino acid residues, and is identical to the region from A723 to R903. Electrophoretic analysis of the lysate prepared from COS-7 cells transfected with Y1- or Y2-expressing vectors gave specific bands corresponding to Y1 or Y2, respectively, showing the validity of the sequences determined. Partial purification of pediatric serum followed by western blotting revealed that Ugl-Y is derived from plasma. Furthermore, Ugl-Y was generated by in vitro digestion of fibronectin by acid protease in extracts of osteoclast cells. These findings suggest that Ugl-Y is probably produced by degradation of fibronectin comprising bone matrix during the process of vigorous bone resorption in children and adolescents. This is the first report on the identification and characterization of juvenile-specific fibronectin fragments excreted into urine.     

Mechanical properties of native and cross-linked type I collagen fibrils

Micromechanical bending experiments using atomic force microscopy were performed to study the mechanical properties of native and carbodiimide-cross-linked single collagen fibrils. Fibrils obtained from a suspension of insoluble collagen type I isolated from bovine Achilles tendon were deposited on a glass substrate containing microchannels. Force-displacement curves recorded at multiple positions along the collagen fibril were used to assess the bending modulus. By fitting the slope of the force-displacement curves recorded at ambient conditions to a model describing the bending of a rod, bending moduli ranging from 1.0 GPa to 3.9 GPa were determined. From a model for anisotropic materials, the shear modulus of the fibril is calculated to be 33 ± 2 MPa at ambient conditions. When fibrils are immersed in phosphate-buffered saline, their bending and shear modulus decrease to 0.07-0.17 GPa and 2.9 ± 0.3 MPa, respectively. The two orders of magnitude lower shear modulus compared with the Young's modulus confirms the mechanical anisotropy of the collagen single fibrils. Cross-linking the collagen fibrils with a water-soluble carbodiimide did not significantly affect the bending modulus. The shear modulus of these fibrils, however, changed to 74 ± 7 MPa at ambient conditions and to 3.4 ± 0.2 MPa in phosphate-buffered saline.     

Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat.

The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.     

Unfolding mechanics of holo- and apocalmodulin studied by the atomic force microscope

The structural stability of calmodulin (CaM) has been investigated previously by chemical and thermal methods. The calcium-loaded form of CaM has been found to be exceptionally stable, because it can be exposed to temperatures >90 degrees C or to a 9 M urea solution without a marked change in its tertiary structure, and is therefore not experimentally accessible for unfolding studies using conventional analytical methods. In this study, we have developed a system for measuring the force for mechanically unfolding CaM using an atomic force microscope (AFM) by stretching the protein from its N- and C-terminal residues; we have been successful in obtaining force versus extension (F-E) curves for both apo and holo forms of CaM. In our experiment, distinguishable F-E curves have been obtained upon stretching of apoCaM and holoCaM to their full extensions. A very low force observed upon stretching of apoCaM indicated a relatively high flexibility of the apo form. On the contrary, a relatively high unfolding force and the appearance of a characteristic force peak were noted during full stretching of holoCaM. The F-E curve of the latter form of CaM most likely reflects a more rigid and probably more organized conformation of holoCaM than that of apoCaM. These experiments confirmed that the AFM is able to clearly distinguish two functionally distinct forms of CaM in terms of their mechanical properties.     

A method for measuring linearly viscoelastic properties of human tympanic membrane using nanoindentation

A viscoelastic nanoindentation technique was developed to measure both in-plane and through-thickness viscoelastic properties of human tympanic membrane (TM). For measurement of in-plane Young's relaxation modulus, the TM sample was clamped on a circular hole and a nanoindenter tip was used to apply a concentrated force at the center of the TM sample. In this setup, the resistance to nanoindentation displacement can be considered due primarily to the in-plane stiffness. The load-displacement curve obtained was used along with finite element analysis to determine the in-plane viscoelastic properties of TM. For measurements of Young's relaxation modulus in the through-thickness (out-of-plane) direction, the TM sample was placed on a relatively rigid solid substrate and nanoindentation was made on the sample surface. In this latter setup, the resistance to nanoindentation displacement arises primarily due to out-of-plane stiffness. The load-displacement curve obtained in this manner was used to determine the out-of-plane relaxation modulus using the method appropriate for viscoelastic materials. From our sample tests, we obtained the steady-state values for in-plane moduli as approximately 17.4 MPa and approximately 19.0 MPa for posterior and anterior portions of TM samples, respectively, and the value for through-thickness modulus as approximately 6.0 MPa for both posterior and anterior TM samples. Using this technique, the local out-of-plane viscoelastic modulus can be determined for different locations over the entire TM, and the in-plane properties can be determined for different quadrants of the TM.     

The acute effect of bipolar radiofrequency energy thermal chondroplasty on intrinsic biomechanical properties and thickness of chondromalacic human articular cartilage

Radio frequency energy (RFE) thermal chondroplasty has been a widely-utilized method of cartilage debridement in the past. Little is known regarding its effect on tissue mechanics. This study investigated the acute biomechanical effects of bipolar RFE treatment on human chondromalacic cartilage. Articular cartilage specimens were extracted (n?=?50) from femoral condyle samples of patients undergoing total knee arthroplasty. Chondromalacia was graded with the Outerbridge classification system. Tissue thicknesses were measured using a needle punch test. Specimens underwent pretreatment load-relaxation testing using a spherical indenter. Bipolar RFE treatment was applied for 45?s and the indentation protocol was repeated. Structural properties were derived from the force-time data. Mechanical properties were derived using a fibril-reinforced biphasic cartilage model. Statistics were performed using repeated measures ANOVA. Cartilage thickness decreased after RFE treatment from a mean of 2.61?mm to 2.20?mm in Grade II, II-III, and III specimens (P?相似文献   

Some structural features of the iron-uptake regulation protein.

An extensive proton nuclear magnetic resonance study of the iron-uptake regulation protein (Fur) from Escherichia coli has been made. Considerable difficulties were experienced in the NMR experiments in 1H2O which may be due unfavourable proton exchange rates in the pH range greater than 6.2, where the protein is soluble. Even in 2H2O, the two-dimensional NMR spectra were not easily interpreted due to widely differing line widths, as a result of the protein side-chains having very differing mobilities. Despite these problems, virtually all the 20 aromatic amino acids have been assigned. Small regions of the protein core were assigned by taking advantage of the approximately 20 non-exchanging peptide-NH resonances in 2H2O. Using two-dimensional J-correlated, homonuclear Hartmann-Hahn and NOE spectroscopies, we have been able to give some assignments in which there is considerable confidence for about one third of the amino acids. Taking advantages of two series of probe experiments, using Mn(II) and a spin label, together with longer range NOE data and result from structure predictions and CD data, we have put forward a tentative fold for the protein which is seen to have a relatively rigid series of interior strands and more flexible exterior strands, many of which are likely to be helical. The Mn(II) probe experiments have also allowed us to define the Fe(II) binding site.     

Elastic modulus of tree frog adhesive toe pads

Previous work using an atomic force microscope in nanoindenter mode indicated that the outer, 10- to 15-??m thick, keratinised layer of tree frog toe pads has a modulus of elasticity equivalent to silicone rubber (5?C15?MPa) (Scholz et al. 2009), but gave no information on the physical properties of deeper structures. In this study, micro-indentation is used to measure the stiffness of whole toe pads of the tree frog, Litoria caerulea. We show here that tree frog toe pads are amongst the softest of biological structures (effective elastic modulus 4?C25?kPa), and that they exhibit a gradient of stiffness, being stiffest on the outside. This stiffness gradient results from the presence of a dense network of capillaries lying beneath the pad epidermis, which probably has a shock absorbing function. Additionally, we compare the physical properties (elastic modulus, work of adhesion, pull-off force) of the toe pads of immature and adult frogs.     

The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP⁺ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.     

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.     

Crystal structure of BstYI at 1.85A resolution: a thermophilic restriction endonuclease with overlapping specificities to BamHI and BglII

We report here the structure of BstYI, an "intermediate" type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5'-RGATCY-3' (where R=A or G and Y=C or T), cleaving DNA after the 5'-R on each strand to produce four-base (5') staggered ends. The crystal structure of free BstYI was solved at 1.85A resolution by multi-wavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the alpha/beta core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional "arm" substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a "relaxed" DNA specificity.     

Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned by various treatments, particularly agitation in aerated or nitrogen-sparged seawater. Stiffness can be permanently increased by more than two times, e.g., from 25 MPa to a maximum of 65 MPa, by simple agitation in aerated seawater. Much but not all of this stiffening can be prevented by agitation under nitrogen. Reversible strain stiffening would seem to be a useful adaptation to lower residual stresses arising from the deformation of two joined materials, i.e., distal and proximal portions with rather different elastic moduli. The permanent strain stiffening that characterizes proximal byssal threads subjected to oxidative stress is probably due to protein cross-linking. In the short term, this results in a stronger thread but at the expense of dynamic interactions between the molecules in the structure.