Hydration and distance dependence of intermolecular shearing between collagen molecules in a model microfibril

In vertebrates, collagen tissues are the main component responsible for force transmission. In spite of the physiological importance of these phenomena, force transmission mechanisms are still not fully understood, especially at smaller scales, including in particular collagen molecules and fibrils. Here we investigate the mechanism of molecular sliding between collagen molecules within a fibril, by shearing a central molecule in a hexagonally packed bundle mimicking the collagen microfibril environment, using varied lateral distance between the molecules in both dry and solvated conditions. In vacuum, the central molecule slides under a stick-slip mechanism that is due to the characteristic surface profile of collagen molecules, enhanced by the breaking and reformation of H-bonds between neighboring collagen molecules. This mechanism is consistently observed for varied lateral separations between molecules. The high shearing force (>7 nN) found for the experimentally observed intermolecular distance (≈1.1 nm) suggests that in dry samples the fibril elongation mechanism relies almost exclusively on molecular stretching, which may explain the higher stiffnesses found in dry fibrils. When hydrated, the slip-stick behavior is observed only below 1.3 nm of lateral distance, whereas above 1.3 nm the molecule shears smoothly, showing that the water layer has a strong lubricating effect. Moreover, the average force required to shear is approximately the same in solvated as in dry conditions (≈2.5 nN), which suggests that the role of water at the intermolecular level includes the transfer of load between molecules.     

Molecular dynamics simulations of a guaiacyl beta-O-4 lignin model compound: examination of intramolecular hydrogen bonding and conformational flexibility

The dynamical conformational behavior of a guaiacyl beta-O-4 lignin model compound has been investigated by molecular simulations. The potential energy surface of the molecule in vacuum has been examined by means of an adiabatic map, showing a large accessible conformational space with multiple energy minima separated by low barriers. Molecular dynamics simulations have been performed in vacuum and with explicit solvent molecules for 10 and 2.1 ns, respectively. Molecular dynamics trajectories recorded in vacuum have shown the molecule to be flexible and to visit a large number of conformations. Many intramolecular H-bonds have been observed, existing for more than 90% of the total simulation time. The presence of explicit solvent molecules induces a significant broadening of some regions of the accessible conformational space and also largely reduces the statistical significance of intramolecular H-bonding. Intramolecular H-bonds observed in vacuum do not persist significantly and are preferentially exchanged with intermolecular H-bonds to the surrounding solvent molecules. The theoretical results are in good agreement with experimental NMR data that do not support the existence of strong and persistent intramolecular H-bonds in solution but instead indicate that H-bonds to solvent predominate. Finally, both molecular modeling and NMR approaches predict the guaiacyl beta-O-4 structure to be flexible and indicate that intramolecular H-bonds are not strong and persistent enough to confer rigidity to the molecule in solution.     

Molecular packing in type I collagen fibrils. A model with neighbouring collagen molecules aligned in axial register

A detailed stereochemical analysis of intermolecular interactions of collagens made with molecular models and summarized experimental data resulted in a new three-dimensional structural model for collagen fibrils. In this model collagen molecules aligned in axial register form a bunch. The bunches are aligned head to tail and penetrate by 300 A into each other, forming microfibrils; these in turn assemble into fibrils. The new model differs from all the others in that its characteristic axial regularity, with a period of 670 A, results from staggering of the adjacent microfibrils formed by unstaggered molecules rather than from the axial staggering of neighbouring collagen molecules.     

The key event in force-induced unfolding of Titin's immunoglobulin domains

Steered molecular dynamics simulation of force-induced titin immunoglobulin domain I27 unfolding led to the discovery of a significant potential energy barrier at an extension of approximately 14 A on the unfolding pathway that protects the domain against stretching. Previous simulations showed that this barrier is due to the concurrent breaking of six interstrand hydrogen bonds (H-bonds) between beta-strands A' and G that is preceded by the breaking of two to three hydrogen bonds between strands A and B, the latter leading to an unfolding intermediate. The simulation results are supported by Angstrom-resolution atomic force microscopy data. Here we perform a structural and energetic analysis of the H-bonds breaking. It is confirmed that H-bonds between strands A and B break rapidly. However, the breaking of the H-bond between strands A' and G needs to be assisted by fluctuations of water molecules. In nanosecond simulations, water molecules are found to repeatedly interact with the protein backbone atoms, weakening individual interstrand H-bonds until all six A'-G H-bonds break simultaneously under the influence of external stretching forces. Only when those bonds are broken can the generic unfolding take place, which involves hydrophobic interactions of the protein core and exerts weaker resistance against stretching than the key event.     

Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.

The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions. Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays. In this study, we have imaged in real-time individual collagen I molecules and their proteolysis by Clostridium histolyticum collagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM). We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules. The proteolysis of collagen molecules was inhibited by reduced calcium and acidification. Results from AFM study of collagen proteolysis are consistent with SDS-PAGE biochemical assays. The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays. This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale. A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level. Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.     

Mechanism of titin unfolding by force: insight from quasi-equilibrium molecular dynamics calculations

We have studied the unfolding by force of one of the immunoglobulin domains of the muscle protein titin using molecular dynamics simulations at 300 K. Previous studies, done at constant pulling rates, showed that under the effect of the force two strands connected to each other by six backbone H-bonds are pulled apart. No details about the mechanism of H-bond breaking were provided. Our simulation protocol "pull and wait" was designed to correspond to very slow pulling, more similar to the rates used in experiments than are the protocols used in previous computational studies. Under these conditions interstrand backbone H-bonds are not "ripped apart" by the application of the force. Instead, small elongations produced by the force weaken specific backbone H-bonds with respect to water-backbone H-bonds. These weakened bonds allow a single water molecule to make H-bonds to the CO and the NH of the same backbone H-bond while they are still bound to each other. The backbone H-bond then breaks (distance > 3.6 A), but its donor and acceptor atoms remain bound to the same water molecule. Further separation of the chains takes place when a second water molecule makes an H-bond with either the protein backbone donor or acceptor atom. Thus, the force does not directly break the main chain H-bonds: it destabilizes them in such a way that they are replaced by H-bonds to water. With this mechanism, the force necessary to break all the H-bonds required to separate the two strands will be strongly dependent on the pulling speed. Further simulations carried out at low forces but long waiting times (> or = 500 ps, < or = 10 ns) show that, given enough time, even a very small pulling force (< 400 pN) is sufficient to destabilize the interstrand H-bonds and allow them to be replaced by H-bonds to two water molecules. As expected, increasing the temperature to 350 K allows the interstrand H-bonds to break at lower forces than those required at 300 K.     

Influence of steroids on hydrogen bonds in membranes assessed by near infrared spectroscopy

The covalent OH bonds of water vibrate and absorb radiation in the near infrared (NIR) region at wavelengths that vary according to the strength of the bonds which, at the same time, are sensitive to the number and/or strength of hydrogen bonds. By means of multivariate analytical tools, such spectral shift was exploited to study the effect of temperature, 25-hydroxycholesterol and progesterone on the H-bonded network of water in DMPA membranes. Temperature was found as the dominating factor altering the NIR spectra of water and then the H-bonds. Increasing temperatures disrupt the H-bonds network, strengthening the OH covalent bonds. The disruption of the H-bonds along the 13–58 °C range was noticeably greater than that caused by lipids or steroids at 500 μM. The H-bonded network of the interfacial water in DMPA membranes was disrupted by the presence of 25-hydroxycholesterol, but no significant disruption was observed in the presence of progesterone. The reduction of the H-bonds entails a reduction in the aggregation of the interfacial water by a reduction in the number of H-bonded molecules. It is proposed that the number of water molecules bonded with two H-bonds diminishes and the number of molecules with no H-bond increases roughly at similar proportions, with a constant population of molecules with one H-bond. The opposed effects of steroids are discussed in the context of their opposed effects on the phase state of membranes, the membrane water content and the steroid molecular structure.     

Unit fibril models derived from the molecular topography of collagen

The geometrical properties of collagen molecules place certain restrictions on possible three-dimensional packing models. With certain reasonable assumptions involving the macromolecular structure of collagen, a model for the surface topography of the monomeric unit is developed and two possible models for molecular packing, one hexagonal and the other non-hexagonal, are inferred. The non-hexagonal packing model is identical to the pentagonal unit fibril first postulated by J. W. Smith on different grounds, while the hexagonal model is one of three previously suggested by us on more general assumptions. The two models are compared to available data on collagen packing, and the bulk of the evidence would seem to favor the pentagonal model.     

Dinosaur peptides suggest mechanisms of protein survival

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.     

Molecular modelling of secondary and tertiary structures of hyaluronan,compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.     

Hydration of protein–RNA recognition sites

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces.     

The type X collagen gene. Intron sequences split the 5'-untranslated region and separate the coding regions for the non-collagenous amino-terminal and triple-helical domains

Type X collagen, expressed by hypertrophic chondrocytes, consists of homotrimeric molecules with subunits that are only about one-half the size of the polypeptides of fibrillar collagens. In this report we describe for the first time the complete primary structure of type X collagen, based on cloning and sequencing of cDNA and genomic DNA. A comparison between the nucleotide sequences of the cDNA and genomic DNA clones has also allowed determination of the complete exon structure of the type X collagen gene. Our results demonstrate that the primary translation product of the chicken type X collagen mRNA is 682 amino acid residues long with a calculated molecular mass of 67,317 Da for the nonhydroxylated form. This calculated molecular mass is in excellent agreement with the observed electrophoretic mobility of cell-free translation products with both poly(A)+ RNA isolated from chondrocytes as well as RNA transcribed in vitro from a full length cDNA construct. It is also in agreement with the observed size of type X collagen polypeptides isolated from the media of cultured hypertrophic chondrocytes. Thus, our data exclude the possibility of a high molecular weight precursor form of type X collagen. Our results also confirm that the chicken type X gene has a most unusual exon structure when compared to other vertebrate collagen genes. The gene has only three exons. One exon (97 base pairs (bp)), codes for most of the 5'-untranslated region of the mRNA, a second exon (159 bp) codes for the signal peptide and a short non-triple-helical domain, while the third exon (2136 bp) contains the coding region for the entire triple-helix and a large non-triple-helical carboxyl domain.     

Critical Main-Chain Length for Conformational Conversion From 3(10)-Helix to α-Helix in Polypeptides

To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.     

Interactions between lignin and urea researched by molecular simulation

Molecular simulations of interactions between urea molecules and lignin polymer have been carried out with the aim of understanding the mechanism of urea slow-release behaviours in lignin–urea materials. It has been found, by docking technology and natural bond orbital analysis, that H-bonds and π-electronic conjugation effect are the main driving forces to keep urea molecules adsorbed on the lignin. In the NPT (isothermal–isobaric ensemble) simulations, mean-squared displacement results show that water molecules can promote the urea molecules gradually away from the lignin. Furthermore, in NVT (canonical ensemble) molecular dynamic simulations, results on diffusion constants of urea molecules in lignin–urea system show that diffusion constant of urea molecules in a urea–water–lignin system increases with an increase in the water content. Conclusions gained from two different kinds of simulation are in agreement with each other and are consistent with the experimental observations.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.     

Protective mechanisms of α,α-trehalose revealed by molecular dynamics simulations

Molecular dynamics simulations were carried out to model aqueous solution with different concentration of α,α-trehalose, one kind of non-reducing sugars possessing outstanding freeze-drying protective effect on biological system. The dihedral angles of the intraglycosidic linkage in trehalose were measured to estimate its structure rigidity. The dynamics and hydrogen bonding properties were studied by calculating the self-diffusion coefficient of trehalose and the distributions and lifetimes of various types of H-bonds in the solution. Through analysing the results as well as comparison with another common sugar sucrose, the freeze-drying protective mechanism of trehalose was explained at molecule level. First, trehalose is able to maintain the local structure around it as a frame due to its relatively rigid conformation. Second, the addition of trehalose restrains the water molecules from rearrangement as a result of low mobility, thus reduces the probability of freezing; trehalose has lower diffusion coefficient than water and bigger thermal diffusivity, which are favourable for vitrification. Third, the formation of H-bonds between trehalose and water and between trehalose molecules is the essence of the protective effect. Trehalose does not work via strengthening the H-bonds formed between water molecules (W–W H-bonds), instead of which it breaks the potential tetrahedral pattern of W–W H-bonds, thus suppresses the tendency of ice formation. It was also found that trehalose realises protective action better at higher concentration as far as this study is concerned.     

The importance of being exhaustive. Optimization of bridging structural water molecules and water networks in models of biological systems

Algorithms and protocols are described for the optimization for H-bonding of isolated singular H2O molecules and entire networks of H2O molecules. Unlike other approaches that are prone to being trapped in local energy minima, these methods rely on exhaustive searches of orientation space for the H2O molecules. The results are scored with the HINT hydropathic interaction model, but the algorithms should be general for any energy-scoring computation. Two examples are provided: 1) the tightly-bound H2O molecule 301 of HIV-1 protease is shown to be more reasonably oriented in terms of forming H-bonds with this method than with a molecular mechanics energy minimization method; and 2) the H2O network surrounding carbonmonoxymyoglobin is constructed and analyzed for a 1.80-A neutron-diffraction structure. The H-atom positions calculated with this method show a somewhat better agreement with the experimental results than do the H-atom positions calculated with molecular mechanics, and both are considerably better than random.     

Physical Models for Exploring DNA Topology

Abstract

Two types of physical models have been developed for treating DNA molecules whose topology is of interest The two model motifs combine jacks-and-straws molecular representations with flexible tubing in different proportions. Both motifs present a low-resolution construct of DNA that retains helix axes, strand individuality and the distinguishabiity of the major and minor grooves. Molecules whose double helix axes are branched are modelled by stiff double helices and flexible branch sites. Supercoiled and knotted DNA molecules are modelled on a smaller scale, in a system in which a flexible backbone is supported by a series of stiff helical struts; removal of this scaffolding immediately reveals the linking of the strands. The models are light and easy to construct. They may be used either for demonstrations or as a research tool that assists the interpretation data.