We present a scanning probe microscope based on optical tweezers for three-dimensional imaging of the topology of transparent material in the nanometer range. A spherical nanoparticle serves as a probe. An optical trap moves it through the sample ͑e.g., a polymer network͒, while the position of the particle center is recorded by three-dimensional interferometry. Accessible volumes are reconstructed from the histogram of thermal position fluctuations of the particle. The resolution in determining the position of surfaces in three dimensions is about 20 nm.

We have used a structure energy-based computer program developed for protein design, Perla, to provide theoretical estimates of all specific side chain-side chain interaction energies occurring in ␣ helices. The computed side chain-side chain interaction energies were used as substitutes for the corresponding values used by the helix/coil transition algorithm, AGADIR. Predictions of peptide helical contents were nearly as successful as those obtained with the originally calibrated set of parameters; a correlation to experimentally observed ␣-helical populations of 0.91 proved that our theoretical estimates are reasonably correct for amino acid pairs that are frequent in our database of peptides. Furthermore, we have determined experimentally the previously uncharacterized interaction energies for Lys-Ile, Thr-Ile, and Phe-Ile amino acid pairs at i,i + 4 positions. The experimental values compare favorably with the computed theoretical estimates. Importantly, the computed values for Thr-Ile and Phe-Ile interactions are better than the energies based on chemical similarity, whereas for Lys-Ile they are similar. Thus, computational techniques can be used to provide precise energies for amino acid pairwise interactions, a fact that supports the development of structure energy-based computational tools for structure predictions and sequence design.Keywords: ␣ Helix; helix/coil transition; peptide design; secondary structure Supplemental material: See www.proteinscience.org.In recent years, enormous progress in the design of small proteins was made. Several computational approaches have been used to successfully design, in an automatic and rational fashion, protein cores (Dahiyat and Mayo 1996;Lazar et al. 1997;Desjarlais and Handel 1995;Lacroix 1999), protein surfaces Lacroix 1999) and a whole protein (Dahiyat and Mayo 1997). To design proteins efficiently, algorithms use a structural, atomic representation to model polypeptide sequences. Amino acid side chains are reconstructed on a defined structural template, and a sequence-structure-stability relationship is established to determine which sequences are advantageous according to a combination of molecular mechanics energy terms and statistical potentials for polypeptide solvation and entropy changes.To test the nature of the energy function, molecular systems simpler than proteins could be used as, for instance, alanine-based ␣-helical peptides. The large amount of data available and the existence of computer programs able to predict the population of folded molecules with considerable accuracy (Lifson and Roig 1961;Muñoz and Serrano 1994a;Chakrabartty and Baldwin 1995;Andersen and Tong 1997;Lomize and Mosberg 1997;Misra and Wong 1997;Lacroix et al. 1998) are advantages supporting the use of helical peptides as models for the precise analysis of interaction energies determined from tridimensional models. An important similarity between computer programs developed for protein design and those used for helical content prediction is the pairwise expression of...

We present a novel experimental method that solves two key problems in nondestructive mechanical studies of small biomolecules at the single-molecule level, namely the confirmation of single-molecule conditions and the discrimination against nonspecific binding. A biotin-avidin ligand-receptor couple is spanned between a glass slide and a 1 microm latex particle using short linker molecules. Optical tweezers are used to initiate bond formation and to follow the particle's thermal position fluctuations with nanometer spatial and microsecond temporal resolution. Here we show that each step in the specific binding process leads to an abrupt change in the magnitude of the particle's thermal position fluctuations, allowing us to count the number of bonds formed one by one. Moreover, three-dimensional position histograms calculated from the particle's fluctuations can be separated into well-defined categories reflecting different binding conditions (single specific, multiple specific, nonspecific). Our method brings quantitative mechanical single-molecule studies to the majority of proteins, paving the way for the investigation of a wide range of phenomena at the single-molecule level.

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