Passive resistance increases differentially in various jaw displacement directions.

OBJECTIVES: In the present study, the passive resistance of the human jaw system was quantified in relation to the three-dimensional jaw displacement and the Posselt-envelope, using both in vivo measurements and computer simulation. METHODS: In eight subjects, the jaw was passively displaced with a step-wise increasing force in three orthogonal directions. Muscle relaxation was monitored using electromyography (EMG) with visual feedback. A biomechanical model of an average human system was used to examine the contributions of the jaw muscles. RESULTS: The largest excursion was found for the vertical direction. Protrusive and lateral directions were more restricted. In protrusive and lateral directions, the jaw could generally move beyond the Posselt-envelope. The stiffness of the jaw increased with proceeding jaw displacement in all directions. The stiffness was larger in the protrusive direction than in the vertical and lateral directions. The model's predictions of stiffness were comparable to the in vivo measurements. However, in protrusive direction, the maximum jaw displacement was larger than in vivo. The estimated passive muscle forces showed that vertical displacement was mainly restricted by the complete group of closing muscles, while protrusive and lateral jaw displacement was restricted by selective individual muscles. CONCLUSIONS: The human jaw system has larger motion range in the protrusive and lateral directions than can be exploited by active muscle use. Stiffness of jaw displacement is higher in the protrusive direction compared to the vertical and lateral directions.

TectoRNA and 'kissing-loop' RNA: atomic force microscopy of self-assembling RNA structures.

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self-assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230-nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a 'kissing-loop' RNA interaction. The second one is a large supramolecular fibre formed of artificial self-assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50-70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.

Sequence-dependent DNA condensation and the electrostatic zipper.

Sequence-dependent configuration changes and condensation of double-stranded poly(dG-dC).(dG-dC) (GC-DNA) and ds poly(dA-dT).(dA-dT) (AT-DNA) were observed by atomic force microscopy in the presence of Ni(II). Less condensing agent was required to generate configuration changes in GC-DNA as compared to AT-DNA. In the presence of Ni(II) cations, GC-DNA adopted a Z-type conformation and underwent a stepwise condensation, starting with partial intramolecular folding, followed by intermolecular condensation of two to several molecules and ending with the formation of toroids, rods, and jumbles. GC-DNA condensates were unusual in that the most highly condensed regions were surrounded by loops of ds GC-DNA. In contrast, AT-DNA retained its B-type conformation and displayed only minor condensation even at high Ni(II) concentrations. The Ni(II)-dependent differences in condensation between GC-DNA and AT-DNA are predicted by an extension of the electrostatic zipper motif proposed by Kornyshev and Leikin, in which we account for shorter than Debye screening length surface separations between the DNA molecules and for the Ni(II)-induced conformation change of GC-DNA to Z-DNA.

Segmented nanofibers of spider dragline silk: atomic force microscopy and single-molecule force spectroscopy.

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

Bone indentation recovery time correlates with bond reforming time.

Despite centuries of work, dating back to Galileo, the molecular basis of bone's toughness and strength remains largely a mystery. A great deal is known about bone microsctructure and the microcracks that are precursors to its fracture, but little is known about the basic mechanism for dissipating the energy of an impact to keep the bone from fracturing. Bone is a nanocomposite of hydroxyapatite crystals and an organic matrix. Because rigid crystals such as the hydroxyapatite crystals cannot dissipate much energy, the organic matrix, which is mainly collagen, must be involved. A reduction in the number of collagen cross links has been associated with reduced bone strength and collagen is molecularly elongated ('pulled') when bovine tendon is strained. Using an atomic force microscope, a molecular mechanistic origin for the remarkable toughness of another biocomposite material, abalone nacre, has been found. Here we report that bone, like abalone nacre, contains polymers with 'sacrificial bonds' that both protect the polymer backbone and dissipate energy. The time needed for these sacrificial bonds to reform after pulling correlates with the time needed for bone to recover its toughness as measured by atomic force microscope indentation testing. We suggest that the sacrificial bonds found within or between collagen molecules may be partially responsible for the toughness of bone.

Surface biology of DNA by atomic force microscopy.

The atomic force microscope operates on surfaces. Since surfaces occupy much of the space in living organisms, surface biology is a valid and valuable form of biology that has been difficult to investigate in the past owing to a lack of good technology. Atomic force microscopy (AFM) of DNA has been used to investigate DNA condensation for gene therapy, DNA mapping and sizing, and a few applications to cancer research and to nanotechnology. Some of the most exciting new applications for atomic force microscopy of DNA involve pulling on single DNA molecules to obtain measurements of single-molecule mechanics and thermodynamics.

Probing biopolymers with the atomic force microscope: a review.

This short review presents an overview of atomic force microscopy (AFM) of biopolymers and specific examples of some of the biopolymers that have been analyzed by AFM. These specific examples include extracellular polymeric substances on the surfaces of bacterial biofilms, condensed DNA, DNA constructs, and DNA-protein interactions. In addition, two examples are presented for AFM analyses of proteins: laminin flexing its arms in solution and neurofilaments entropically brushing away the space around themselves.

Probing protein-protein interactions in real time.

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.

Basement membrane macromolecules: insights from atomic force microscopy.

The major macromolecules of basement membranes-collagen IV, laminin-1, and heparan sulfate proteoglycan (HSPG)-have been analyzed by atomic force microscopy (AFM), both individually and in combination with each other. The positions of laminin binding to collagen IV were mapped and compared with the positions of imperfections in the amino acid sequence of collagen IV; the apparent molecular volumes of the HSPG proteoglycans were measured and used to estimate the corresponding molecular weights. Even the thin, thread-like strands of the polyanion heparan sulfate can be visualized with AFM without staining, coating, or fixation. These strands are single polysaccharide chains and are thus thinner than single-stranded DNA. The heparan sulfate strands in HSPG are necessary for protein filtration in kidney basement membranes. We propose that these thin strands filter proteins by functioning as an entropic brush-i.e., that they filter proteins by their constant thermally driven motion in the basement membrane. These AFM analyses in air are a step toward AFM analyses under fluid of basement membrane macromolecules interacting with each other.

Physical morphology and surface properties of unsaturated Pseudomonas putida biofilms.

Unsaturated biofilms of Pseudomonas putida, i.e., biofilms grown in humid air, were analyzed by atomic force microscopy to determine surface morphology, roughness, and adhesion forces in the outer and basal cell layers of fresh and desiccated biofilms. Desiccated biofilms were equilibrated with a 75.5% relative humidity atmosphere, which is far below the relative humidity of 98 to 99% at which these biofilms were cultured. In sharp contrast to the effects of drying on biofilms grown in fluid, we observed that drying caused little change in morphology, roughness, or adhesion forces in these unsaturated biofilms. Surface roughness for moist and dry biofilms increased approximately linearly with increasing scan sizes. This indicated that the divides between bacteria contributed more to overall roughness than did extracellular polymeric substances (EPS) on individual bacteria. The EPS formed higher-order structures we termed mesostructures. These mesostructures are much larger than the discrete polymers of glycolipids and proteins that have been previously characterized on the outer surface of these gram-negative bacteria.

Recent highlights from atomic force microscopy of DNA.

Abstract Seven recent highlights are presented from atomic force microscopy (AFM) of DNA in this lab. The first two involve advances in the observation of enzymatic reactions in near-physiological solutions. E. coli RNA polymerase was observed to process along its DNA template in a series of time-lapse images [S. Kasas, et al., Biochemistry 36, 461 (1997)], and a new small-cantilever atomic force microscope (AFM) imaged DNA degradation by DNase I at rates as fast as two seconds per image. The next five highlights involve structural observations of DNA and DNA-protein complexes, including DNA condensed for gene delivery, sequence-dependent DNA condensation, an AFM assay for RNA polymerase, and AFM evidence for a yeast kinetochore complex that may be involved in holding together sister chromatids during cell division.

Polymerase activities and RNA structures in the atomic force microscope.

The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA. Rapid semiquantitative estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme concatamer and an RNA with 90% purines.

Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy.

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

Atomic force microscopy and other scanning probe microscopies.

The highlight of the past year is the unfolding and refolding of the muscle protein titin in the atomic force microscope. A related highlight in the intersection between experiment and theory is a recent review of the effects of molecular forces on biochemical kinetics. Other advances in scanning probe microscopy include entropic brushes, molecular sandwiches and applications of atomic force microscopy to gene therapy.

Structures and dynamic motion of laminin-1 as observed by atomic force microscopy.

Laminins are a family of multifunctional extracellular matrix glycoproteins that play important roles in the development and maintenance of tissue organization via their interactions with cells and other extracellular matrix proteins. To understand the structural basis of laminins' functions, we examined the motion of laminin-1 (Ln-1) in physiological buffers using atomic force microscopy. While many Ln-1 molecules assumed the expected cruciform structure, unexpected dynamic movements of the Ln-1 arms were observed in aqueous environments. These dynamic movements of the Ln-1 arms may contribute to the diversity of laminin functions.

DNA condensation for gene therapy as monitored by atomic force microscopy.

The atomic force microscope (AFM) was used to assay the extent of DNA condensation in approximately 100 different complexes of DNA with polylysine (PL) or PL covalently attached to the glycoproteins asialoorosomucoid (AsOR) or orosomucoid (OR). The best condensation of DNA was obtained with 10 kDa PL covalently attached to AsOR, at a lysine:nucleotide (Lys:nt) ratio of 5:1 or higher. These conditions produce large numbers of toroids and short rods with contour lengths of 300-400 nm. Some DNA condensation into shortened thickened structures was seen with 10 kDa PL attached to AsOR at Lys:nt ratios of 1.6:1 and 3:1. Some DNA condensation was also seen with 4 kDa PL at Lys:nt ratios of 3:1 and higher. Little DNA condensation was seen with PL alone or with PL convalently attached to OR at Lys:nt ratios up to 6:1. AsOR-PL enhanced gene expression in the mouse liver approximately 10- to 50-fold as compared with PL alone.

Substructure and responses of cholinergic synaptic vesicles in the atomic force microscope.

The substructure and responses of individual 100-nm synaptic vesicles to osmotic stress have been probed with an atomic force microscope (AFM) operating in tapping mode. Cholinergic synaptic vesicles from the electric organ of Torpedo californica were imaged continuously as the osmolarity of the buffer was decreased. Vesicles in hyposmotic buffer lysed to form flat circular structures on the mica surface with a diameter about two times that of intact vesicles and a thickness of 7.2 +/- 1.7 nm, which can accommodate the lipid bilayer plus the internal proteoglycan. Images of intact vesicles in air reveal creases in the membrane surface. Phase mode AFM images of lysed vesicles in air show the presence of a material not seen on intact vesicles that might be intravesicular proteoglycan released from the membrane at very low osmotic and ionic strength.