ScienceWeek

BIOPHYSICS: SCANNING PROBE MICROSCOPY IN BIOLOGY

The following points are made by J. K. H. Hoerber and M. J. Miles (Science 2003 302:1002):

1) The importance of the development of scanning probe microscopy (SPM) is comparable to that of electron microscopy and even optical microscopy. SPM measures the near-field physical interactions between the scanning probe tip and the atoms that lie beneath it as it moves. Atomic force microscopy (AFM) (1), a member of the SPM family, has become particularly important for the study of biological systems. AFM produces three-dimensional (3D) images of a surface at atomic resolution; even subatomic resolution has been reported for atomic orbitals on a silicon surface (2). Its major advantage is that it can produce high-resolution topographic images in aqueous and physiologically relevant environments without the need to stain the specimen; the AFM contrast mechanism does not depend on atomic number, but simply senses the specimen surface through the force between it and a sharp probe that scans the surface.

2) Local probes, the central feature of all scanning probe instruments, are small-sized objects such as the ends of sharp tips; the interaction of the tip with the surface of a sample can be sensed at selected positions. Proximity to or contact with the sample is required for high spatial resolution. The principal idea is quite old and had appeared in literature from time to time, in the context of bringing a source of electromagnetic radiation in close contact with a sample, but was not pursued until recently. Nanoscale local probes require atomically stable tips and high-precision manipulation devices. The latter are based on mechanical deformations of springlike structures by given forces -- piezoelectric, mechanical, electrostatic, or magnetic -- to ensure continuous and reproducible displacements with precision down to the picometer level. They also require effective acoustic, thermal, and vibration isolation. The resolution that can be achieved with local probes is primarily a function of the effective probe size, its distance from the sample, and the distance dependence of the interaction between probe and sample measured. The last can be considered to create an effective aperture by selecting a small feature of the overall geometry of the probe tip, which then corresponds to the effective probe.

3) The ability of AFM to yield images under ambient conditions or in solution was considered from the outset to provide an ideal tool for physical studies of biological specimens under physiological conditions. Contact-mode, constant-force AFM imaging could show processes induced by viral infection on live cells (3) and has achieved atomic-scale resolution images of cellulose microfibrils (4). In recent years, high-resolution AFM imaging has also been achieved on bacterial membrane proteins (5). Recently, the analysis of AFM images of 2D crystals of membrane proteins such as aquaporin-Z has been developed to the extent that the free energy landscape can be derived for domains within single protein molecules. From the raw AFM data, an average topograph for a single protein molecule is calculated with processing techniques more commonly used in transmission electron microscopy. From this, the conformational space of the protein can be derived by calculating the standard deviation from this average structure of all the protein topographs. Similarly, the probability distribution of topographic peaks in the protein domains under thermal motion can be calculated, and from this the free energy landscape can be derived. A comparison of this landscape can then be correlated with the atomic structure of the protein.

4) In summary: Twenty years ago the first scanning probe instrument, the scanning tunneling microscope, opened up new realms for our perception of the world. Atoms that had been abstract entities were now real objects, clearly seen as distinguishable individuals at particular positions in space. A whole family of scanning probe instruments has been developed, extending our sense of touching to the scale of atoms and molecules. Such instruments are especially useful for imaging of biomolecular structures because they can produce topographic images with submolecular resolution in aqueous environments. Instruments with increased imaging rates, lower probe-specimen force interactions, and probe configurations not constrained to planar surfaces are being developed, with the goal of imaging processes at the single-molecule level -- not only at surfaces but also within three-dimensional volumes—in real time.

References (abridged):

1. G. Binnig, C. F. Quate, C. Gerber, Phys. Rev. Lett. 56, 930(1986)

2. F. J. Giessibl, Science 267, 68 (1995)

3. W. Häberle, J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, G. Binnig, Ultramicroscopy 42-44, 1161 (1992)

4. A. A. Baker, W. Helbert, J. Sugiyama, M. J. Miles, Biophys. J. 79, 1139 (2000)

5. S. Scheuring, D. J. Mueller, H. Stahlberg, H.-A. Engel, A. Engel, Eur. Biophys. J. 31, 172 (2002)

Science http://www.sciencemag.org

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REVERSE ENGINEERING OF THE GIANT MUSCLE PROTEIN TITIN

The term "myofibril" refers to one of the long cylindrical contractile elements that constitute the major component of the muscle fiber, extending for the entire length of the muscle fiber, with each myofibril composed in turn of numerous myofilaments.

The term "sarcomere" refers to the functional unit of a myofibril of vertebrate muscle, each sarcomere approximately 2 to 3 microns long. In striated muscle, the sarcomeres of many parallel myofibrils are positioned such that myosin thick filaments are aligned in register across the myofibril and have a dense appearance in microscope preparations. The actin thin filaments have a lighter appearance, with a resulting alteration of light isotropic bands called "I bands" and dark anisotropic bands called "A bands". The I band is bisected by a dense narrow "Z line", while the central less dense region of the A band is known as the "H zone", which in turn is bisected by the dark "M line" (or midline), the locus of specific proteins that link adjacent thick filaments to each other. The Z lines are due to attachment sites for thin filaments. The various sarcomere bands are thus classical light-microscope features which have only in the past few decades been analyzed in terms of various macromolecular structures.

The term "titin" (connectin) refers to a giant protein that forms a single-molecule elastic filament extending from the M line to the Z line in the striated muscle sarcomere. Titin is one of the largest polypeptides yet described. Its amino acid sequence consists mainly of repeats of two types of approximately 100-amino-acid motifs, known as class I and class II. There is also a domain characteristic of protein kinases near the C terminus. Titin is believed to play an important role in sarcomere alignment during muscle contraction. A single molecule of titin measures approximately 1 micron in length and has a molecular weight of approximately 3 million daltons.

The following points are made by H. Li et al (Nature 2002 418:998):

1) Individual titin molecules span both the A-band and I-band regions of muscle sarcomeres. The I-band part of titin has been identified as the region that is functionally elastic. The authors report a study of the shortest titin isoform, the N2B isoform found in cardiac-muscle sarcomeres. The elastic I-band region of N2B-titin can be subdivided into four structurally distinct regions: a proximal immunoglobulin region containing 15 tandem immunoglobulin-like (Ig) domains; a middle N2B segment that contains a 572-residue amino-acid sequence of unknown structure; a 186-amino-acid-long segment rich in proline (P), glutamate (E), valine (V) and lysine (K) residues, named the PEVK region; and a distal Ig region that contains 22 tandem Ig modules. The authors use polyprotein engineering and single-molecule force spectroscopy to dissect the individual mechanical elements of the I-band of cardiac titin and reconstruct the elasticity of cardiac muscle. Polyproteins, when mechanically stretched by single-molecule atomic force microscopy (AFM) give distinctive mechanical fingerprints as their modules unfold sequentially (sawtooth patterns in the force extension curve), and can be used to positively identify the mechanical features of a single molecule.

2) In summary: Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells(1-5). The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. The authors use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. The authors demonstrate that when these mechanical elements are combined, they explain the macroscopic behavior of titin in intact muscle. The authors suggest their studies demonstrate the functional reconstitution of a protein from the sum of its parts.

References (abridged):

1. Sigworth, F. J. & Neher, E. Single Na+ channel currents observed in cultured rat muscle cells. Nature 287, 447-449 (1980)

2. Bustamante, C., Smith, S. B., Liphardt, J. & Smith, D. Single-molecule studies of DNA mechanics. Curr. Opin. Struct. Biol. 10, 279-285 (2000)

3. Smith, D. E. et al. The bacteriophage 29 portal motor can package DNA against a large internal force. Nature 413, 748-752 (2001)

4. Finer, J. T., Simmons, R. M. & Spudich, J. A. Single myosin molecule mechanics: piconewton forces and nanometer steps. Nature 368, 113-119 (1994)

5. Lu, H. & Schulten, K. Steered molecular dynamics simulations of force-induced protein domain unfolding. Proteins Struct. Funct. Genet. 35, 453-463 (1999)

Nature http://www.nature.com/nature

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