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Biomedical subjects

M Rief

Publications and source records attributed to M Rief.

14 recordsLinked to original sources
PubMed2005

Influence of substrate binding on the mechanical stability of mouse dihydrofolate reductase.

We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of approximately 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.

Animals↗
PubMed2000

Myosin-V stepping kinetics: a molecular model for processivity.

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

Actins↗
PubMed2000

In vitro assays of processive myosin motors.

Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.

Actin Cytoskeleton↗
PubMed2000

Unfolding forces of titin and fibronectin domains directly measured by AFM.

AFM-based Single Molecule Force Spectroscopy provides a new tool for probing the mechanical properties of single molecules. In this chapter we show that the unfolding forces of single protein domains can be directly measured. Unfolding forces give new insight into protein stability that cannot be deduced from thermodynamic measurements. A comparison between the unfolding forces measured in Ig domains of the muscle protein titin and those measured in fibronectin Type III domains reveals an extraordinarily high stability of titin domains.

Connectin↗
PubMed2000

Mechanical stability of single DNA molecules.

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (</=10 mM NaCl), while high ionic strength buffers (1 M NaCl) stabilize the double-helical conformation. The mechanical energy that can be deposited in the DNA double helix before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with the base-pairing free enthalpy DeltaG(bp)(T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences again revealed a close correlation between the mechanical energies at which these sequences melt with base pairing free enthalpies DeltaG(bp)(sequence): while the melting transition occurs between 65 and 200 pN in lambda-phage DNA, depending on the loading rate, the melting transition is shifted to approximately 300 pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA melts at a force of 35 pN.

Bacteriophage lambda↗
PubMed1999

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

Actins↗
PubMed1999

Single-molecule biomechanics with optical methods.

Single-molecule observation and manipulation have come of age. With the advent of optical tweezers and other methods for probing and imaging single molecules, investigators have circumvented the model-dependent extrapolation from ensemble assays that has been the hallmark of classical biochemistry and biophysics. In recent years, there have been important advances in the understanding of how motor proteins work. The range of these technologies has also started to expand into areas such as DNA transcription and protein folding. Here, recent experiments with rotary motors, linear motors, RNA polymerase, and titin are described.

Biomechanical Phenomena↗
PubMed1999

How strong is a covalent bond?

The rupture force of single covalent bonds under an external load was measured with an atomic force microscope (AFM). Single polysaccharide molecules were covalently anchored between a surface and an AFM tip and then stretched until they became detached. By using different surface chemistries for the attachment, it was found that the silicon-carbon bond ruptured at 2.0 +/- 0.3 nanonewtons, whereas the sulfur-gold anchor ruptured at 1.4 +/- 0.3 nanonewtons at force-loading rates of 10 nanonewtons per second. Bond rupture probability calculations that were based on density functional theory corroborate the measured values.

Journal Article↗
PubMed1999

Single molecule force spectroscopy of spectrin repeats: low unfolding forces in helix bundles.

Spectrin repeats fold into triple helical coiled-coils comprising approximately 106 amino acid residues. Using an AFM-related technique we measured the force required to mechanically unfold these repeats to be 25 to 35 pN. Under tension, individual spectrin repeats unfold independently and in an all-or-none process. The dependence of the unfolding forces on the pulling speed reveals that the corresponding unfolding potential is shallow with an estimated width of 1.5 nm. When the unfolded polypeptide strand is relaxed, several domains refold within less than a second. The unfolding forces of the alpha-helical spectrin domains are five to ten times lower than those found in domains with beta-fold, like immunoglobulin or fibronectin Ill domains, where the tertiary structure is stabilized by hydrogen bonds between adjacent strands. This shows that the forces stabilizing the coiled-coil lead to a mechanically much weaker structure than multiple hydrogen-bonded beta-sheets.

Animals↗
PubMed1999

Sequence-dependent mechanics of single DNA molecules.

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.

Bacteriophage lambda↗
PubMed1998

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).

Biomechanical Phenomena↗
PubMed1997

Reversible unfolding of individual titin immunoglobulin domains by AFM.

Single-molecule atomic force microscopy (AFM) was used to investigate the mechanical properties of titin, the giant sarcomeric protein of striated muscle. Individual titin molecules were repeatedly stretched, and the applied force was recorded as a function of the elongation. At large extensions, the restoring force exhibited a sawtoothlike pattern, with a periodicity that varied between 25 and 28 nanometers. Measurements of recombinant titin immunoglobulin segments of two different lengths exhibited the same pattern and allowed attribution of the discontinuities to the unfolding of individual immunoglobulin domains. The forces required to unfold individual domains ranged from 150 to 300 piconewtons and depended on the pulling speed. Upon relaxation, refolding of immunoglobulin domains was observed.

Adsorption↗
PubMed1997

Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy

Recent developments in piconewton instrumentation allow the manipulation of single molecules and measurements of intermolecular as well as intramolecular forces. Dextran filaments linked to a gold surface were probed with the atomic force microscope tip by vertical stretching. At low forces the deformation of dextran was found to be dominated by entropic forces and can be described by the Langevin function with a 6 angstrom Kuhn length. At elevated forces the strand elongation was governed by a twist of bond angles. At higher forces the dextran filaments underwent a distinct conformational change. The polymer stiffened and the segment elasticity was dominated by the bending of bond angles. The conformational change was found to be reversible and was corroborated by molecular dynamics calculations.

Journal Article↗