Relationship between functional properties and structure of ovalbumin

The effects of ovalbumin (OVA) denaturation using urea, guanidinium chloride (GdnHCl), sodium dodecyl sulphate (SDS), cetylpyridinium chloride (CPC), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), and 5 different cationic detergents with various side chains, HCl, and CH₃COOH were observed. Progressive unfolding in ovalbumin was measured as a function of fluorescent light intensity, peak response and shift in the maximum of emission. Kinetic measurements demonstrated that the rate of denaturation usually followed a double exponential decay pattern, but at small concentrations of urea and acids first-order reaction was indicated. The reversibility of the unfolding-folding transitions was confirmed from tryptophan fluorescence and circular dichroism (CD) measurements. Differences in secondary structure were observed and changes of-helical content were calculated. Polyacrylamide gel electrophoresis (PAGE) with and without sodium dodecyl sulphate (SDS-PAGE) showed differences in the structure of native and denatured ovalbumin. Native protein samples in PAGE demonstrated smaller number and larger mobilities of subunits than denatured ones with different reductants, such as SDS and 2-mercaptoethanol (2 ME). Scanning of SDS protein patterns showed the appearance of aggregated forms in region of 45 kD.     

Relationship between triphasic mechanical properties of articular cartilage and osteoarthritic grade

The purpose of this study was to explore the triphasic mechanical properties of osteoarthritic cartilage with different pathological grades. First, samples of cartilage from rabbits with different stages of osteoarthritis (OA) were graded. Following this, the cartilage was strained by a swelling experiment, and changes were measured using a high-frequency ultrasound system. The result, together with fixed charge density and water volume fraction of cartilage samples, was used to estimate the uniaxial modulus of the cartilage tissue, based on a triphasic model. For the control cartilage samples, the uniaxial elastic modulus on the cartilage surface was lower than those in the middle and deep layers. With an increase in the OA grade, the uniaxial elastic modulus of the surface, middle and deep layers decreased. A significant difference was found in the surface elastic modulus of different OA grades (P<0.01), while no significant differences were identified for OA cartilages of Grades 1 and 2 in the middle and deep layers (P<0.01). Compared with Grades 1 and 2, there was a significant reduction in the elastic modulus in the middle and deep layers of Grade 3 OA cartilage (P<0.05). Overall, this study may provide a new quantitative method to evaluate the severity of OA using the mechanical properties of cartilage tissue.     

Influence of chondroitin sulfate on collagen gel structure and mechanical properties at physiologically relevant levels

The ability to alter collagen organization could lead to more physiologically relevant scaffolds for tissue engineering. This study examined collagen organization in the presence of polysaccharide and the resulting effects on viscoelastic properties. Fibrillogenesis in the presence of chondroitin sulfate (CS) resulted in changes in the collagen network organization with an increase in void space present. The increased void space caused by CS addition correlated with a decreased stiffness of the collagen gel. These changes occurred with physiologically relevant ratios of collagen to CS, at physiological pH and ionic strength, and without a decrease in the amount of collagen incorporated into fibrils. The addition of dextran, an uncharged polysaccharide, yielded no change in network void space or mechanical properties. Changes in fibril diameter caused by CS or dextran were not correlated with mechanical properties. The results of this study demonstrate that collagen organization can be modified by the addition of GAG, leading to altered matrix mechanical properties. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 841-851, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Age-related thermodynamic and mechanical properties of the rat collagen

The age peculiarities of cross-linking degree, thermodynamic and mechanic properties of rat tail tendon collagen fibres were investigated. It is shown that during the period from 1 to 3 months the melting temperature decreases and the enthalpy difference increases, from 3 to 24 months the melting temperature increases and the enthalpy difference decreases. The strength of fibres increases during the whole life. The maximal relative extension increases during the first 12 months and tends to decrease in after-life. The Young's module in the elastic deformation region decreases during the period from 1 to 3 months, then increases. It is shown tht those changes in fibre properties may be connected with the age dynamics of collagen cross-linking degree observed here: its decreasing during the period from 1 to 3 months and its following continuous increasing in after-life.     

The molecular and fibrillar structure of collagen and its relationship to the mechanical properties of connective tissue

The conformation of type I collagen molecules has been refined using a linked-atom least-squares procedure in conjunction with high-quality X-ray diffraction data. In many tendons these molecules pack in crystalline arrays and a careful measurement of the positions of the Bragg reflections allows the unit cell to be determined with high precision. From a further analysis of the X-ray data it can be shown that the highly ordered overlap region of the collagen fibrils consists of a crystalline array of molecular segments inclined by a small angle with respect to the fibril axis. In contrast, the gap region is less well ordered and contains molecular segments that are likely to be inclined by a similar angle but in a different vertical plane to that found in the overlap region. The collagen molecule thus has a D-periodic crimp in addition to the macroscopic crimp observed visually in the collagen fibres of many connective tissues. The growth and development of collagen fibrils have been studied by electron microscopy for a diverse range of connective tissues and the general pattern of fibril growth has been established as a function of age. In particular, relationships between fibril size distribution, the content and composition of the glycosaminoglycans in the matrix and the mechanical role played by the fibrils in the tissue have been formulated and these now seem capable of explaining many new facets of connective tissue structure and function.     

Cooperative transition between open and closed conformations in potassium channels

Potassium (K+) ion channels switch between open and closed conformations. The nature of this important transition was revealed by comparing the X-ray crystal structures of the MthK channel from Methanobacterium thermoautotrophicum, obtained in its open conformation, and the KcsA channel from Streptomyces lividans, obtained in its closed conformation. We analyzed the dynamic characteristics and energetics of these homotetrameric structures in order to study the role of the intersubunit cooperativity in this transition. For this, elastic models and in silico alanine-scanning mutagenesis were used, respectively. Reassuringly, the calculations manifested motion from the open (closed) towards the closed (open) conformation. The calculations also revealed a network of dynamically and energetically coupled residues. Interestingly, the network suggests coupling between the selectivity filter and the gate, which are located at the two ends of the channel pore. Coupling between these two regions was not observed in calculations that were conducted with the monomer, which emphasizes the importance of the intersubunit interactions within the tetrameric structure for the cooperative gating behavior of the channel.     

The relation between collagen fibril kinematics and mechanical properties in the mitral valve anterior leaflet

We have recently demonstrated that the mitral valve anterior leaflet (MVAL) exhibited minimal hysteresis, no strain rate sensitivity, stress relaxation but not creep (Grashow et al., 2006, Ann Biomed Eng., 34(2), pp. 315-325; Grashow et al., 2006, Ann Biomed. Eng., 34(10), pp. 1509-1518). However, the underlying structural basis for this unique quasi-elastic mechanical behavior is presently unknown. As collagen is the major structural component of the MVAL, we investigated the relation between collagen fibril kinematics (rotation and stretch) and tissue-level mechanical properties in the MVAL under biaxial loading using small angle X-ray scattering. A novel device was developed and utilized to perform simultaneous measurements of tissue level forces and strain under a planar biaxial loading state. Collagen fibril D-period strain (epsilonD) and the fibrillar angular distribution were measured under equibiaxial tension, creep, and stress relaxation to a peak tension of 90 N/m. Results indicated that, under equibiaxial tension, collagen fibril straining did not initiate until the end of the nonlinear region of the tissue-level stress-strain curve. At higher tissue tension levels, epsilonD increased linearly with increasing tension. Changes in the angular distribution of the collagen fibrils mainly occurred in the tissue toe region. Using epsilonD, the tangent modulus of collagen fibrils was estimated to be 95.5+/-25.5 MPa, which was approximately 27 times higher than the tissue tensile tangent modulus of 3.58+/-1.83 MPa. In creep tests performed at 90 N/m equibiaxial tension for 60 min, both tissue strain and epsilonD remained constant with no observable changes over the test length. In contrast, in stress relaxation tests performed for 90 min epsilonD was found to rapidly decrease in the first 10 min followed by a slower decay rate for the remainder of the test. Using a single exponential model, the time constant for the reduction in collagen fibril strain was 8.3 min, which was smaller than the tissue-level stress relaxation time constants of 22.0 and 16.9 min in the circumferential and radial directions, respectively. Moreover, there was no change in the fibril angular distribution under both creep and stress relaxation over the test period. Our results suggest that (1) the MVAL collagen fibrils do not exhibit intrinsic viscoelastic behavior, (2) tissue relaxation results from the removal of stress from the fibrils, possibly by a slipping mechanism modulated by noncollagenous components (e.g. proteoglycans), and (3) the lack of creep but the occurrence of stress relaxation suggests a "load-locking" behavior under maintained loading conditions. These unique mechanical characteristics are likely necessary for normal valvular function.     

Discrimination between closed and open forms of lipases using electrophoretic techniques

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.     

Calculations of quantum tunnelling between closed and open states of sodium channels

Transitions between states of ion channels have previously been considered in terms of classical statistical mechanics. However, transitions in many systems, including some organic molecules, are known to occur by quantum mechanical tunnelling. In this report, we have calculated the time for sodium channel activation by tunnelling, starting from a mechanistic model based on the structural models of Catterall and Guy. In doing this, we have calculated the Coulomb interactions between the S4 -helix and negative residues on nearest-neighbor helices and have included longer range interactions in terms of an effective background interaction. Periodic pairing of charges between the S4 and adjacent helices in the model causes the resting and depolarized states of the channel to correspond to local minima in the S4 potential energy curve. Harmonic potentials closely fit the energy curves around each of the two minima and the energy barrier between them is closely modelled by a parabola. These approximations allow a semiclassical calculation of the S4 helix's tunnelling rate to be made. At 37°C, for an interhelix axial spacing of 10 Å, tunnelling times in the range of 1 s to a few ms were computed for a single S4 segment, depending of the equilibrium temperature of the cell membrane.     

Relationship between collagen fibril diameters and body size

Summary Suspensions of collagen fibrils obtained from derma of Elasmobranchia and Actinopterygia of different body sizes and developmental stages were examined by transmission electron microscopy. Fibril diameters were measured and classified into groups comprising a 20 nm diameter interval. Diagrams showing fibril populations of each fish were made. The measurements were averaged and their confidence intervals and standard errors determined. For each species other diagrams were plotted in which the mean diameters were correlated to the body length of each sample. The results show that: 1) a correlation exists between an increase in diameter of collagen fibrils and somatic growth until sexual maturity is reached; 2) fibril populations are subsequently spread over a wider range due to the presence in the derma of classes of newly formed and therefore thinner fibrils. The deposition of new fibrils is possibly influenced by individual factors; 3) no relationship exists between mean fibril diameter and body size; 4) no relationship exists between phylogenetic position and pattern of diameter distribution.Research supported by a grant from C.N.R. Roma (69.02087.0115.1150)     

Changes in collagen structure under the influence of mechanical dispersion

Air-dry collagen isolated from cattle retinal layer by means of alkaline-salt treatment was crushed in a laboratory vibro-mill at 80-150 degrees K. Mechanochemical transformations were studied by means of viscosimetry, polarimetry, ESR-spectroscopy and electron microscopy. Mechanical tensions induce breakage of covalent bonds of polypeptide chains, accompanied by a decrease of protein molecular mass, and of lateral interactions, which results in loosening of collagen structure and partial denaturation.     

EEG differences between resting states with eyes open and closed in darkness

Transition from a resting state with eyes closed (REC) to a resting state with eyes open (REO) is associated with visible changes in EEG, which are traditionally considered to be a sign of reorganization of the brain’s activity in response to visual stimuli. The EEGs recorded in the REC and REO states in complete darkness, when the stimulatory effect of light to the eye’s retina was absent, were compared. Thirty healthy subjects participated in the study. EEG in the range of 1.5–50 Hz was recorded from nineteen zones of the head monopolarly. It was found that, under conditions of complete darkness, the REC and REO states significantly differed in their EEG spectral power and coherence in the Δ, θ, α₁, α₂, β₁, β₂ and γ frequency bands. Under experimental conditions, these changes in the EEG could not be induced by external influence to the visual system. Therefore, we suppose that they are correlates of the switching of involuntary preliminary attention from internally directed attention specific for the REC state to externally directed attention specific for the REO state.     

Simultaneous processing of fibril formation and cross-linking improves mechanical properties of collagen

In vitro "simultaneous processing" was investigated in which fibril formation of collagen and cross-linking occur simultaneously in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as a cross-linking reagent. Fibril formation in simultaneous processing was monitored using turbidity. The EDC in simultaneous processing increased T(1/2) (time required for half of the plateau value in turbidity) and decreased the degree of the fibril formation dose dependently. The reduced fibril formation rate (T(1/2) > 60 s) suggests the introduction of intrafibrillar cross-linking during fibril formation. The collagen gels prepared using simultaneous processing had a compressive modulus that was 6-fold higher than that using sequential processing, which is an advantage of simultaneous processing. Atomic force microscopy images acquired under water on the wet gels demonstrated that the simultaneous processing provided a unique double-network structure: intrafibrillarly cross-linked collagen fibrils among which nonfibrous collagens act as interfibrillar cross-linkages.     

One channel: open and closed

Structural information about the prokaryotic KirBac3.1 inward rectifier family K(+) channel from Magnetospirillum magnetotacticum is reported. These results from two-dimensional electron cryomicroscopy (EM) shed light on the gating mechanism of members of the Kir channel family.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.