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.

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.

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Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.

The combination of high-resolution atomic force microscopy imaging and single-molecule force spectroscopy allows the identification, selection, and mechanical investigation of individual proteins. In a recent paper we had used this technique to unfold and extract single bacteriorhodopsins (BRs) from native purple membrane patches. We show that subsets of the unfolding spectra can be classified and grouped to reveal detailed insight into the individualism of the unfolding pathways. We have further developed this technique and analysis to report here on the influence of pH effects and local mutations on the stability of individual structural elements of BR against mechanical unfolding. We found that, although the seven transmembrane alpha-helices predominantly unfold in pairs, each of the helices may also unfold individually and in some cases even only partially. Additionally, intermittent states in the unfolding process were found, which are associated with the stretching of the extracellular loops connecting the alpha-helices. This suggests that polypeptide loops potentially act as a barrier to unfolding and contribute significantly to the structural stability of BR. Chemical removal of the Schiff base, the covalent linkage of the photoactive retinal to the helix G, resulted in a predominantly two-step unfolding of this helix. It is concluded that the covalent linkage of the retinal to helix G stabilizes the structure of BR. Trapping mutant D96N in the M state of the proton pumping photocycle did not affect the unfolding barriers of BR.

We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein-or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16°. For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32°؎ 6°, 56°؎ 4°a nd 73 ؎ 2°, respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.absolute distance measurements ͉ fluorescence energy transfer ͉ multiparameter fluorescence detection ͉ nucleic acid structures I n recent years, fluorescence energy transfer experiments (FRET) have shown great potential for subnanometer analysis of biomolecular structures and their dynamics that can even be applied to single molecules (1-5). Calculation of absolute FRET distances between a donor and acceptor fluorophore is complicated because of several ''calibration'' factors such as detection efficiencies, spectral cross-talk and fluorescence quantum yields (6) that are difficult to determine accurately. Multiparameter Fluorescence Detection (MFD) (7) avoids most pitfalls (5) by simultaneously collecting all fluorescence parameters (intensity, lifetime and anisotropy in both spectral ranges) at the singlemolecule level.However, determination of absolute distances remains a major challenge because of the uncertainty in the fluorophore positions. This uncertainty is due to the use of the long linkers through which the fluorophores are attached to the biomolecules. Orientational freedom is a prerequisite to safely assume an orientation factor ( 2 ) of 2/3 (8); however, this prevents a defined fluorophore position, which is needed for fitting data to a geometric model. Here, we present a new conversion function for experimental FRET efficiencies that considers the dynamics of the movement of the fluorop...

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