Inspired by Biology From Molecules to Materials to Machines (2008) / Chapter Skim
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4 Probes and Tools for Biomolecular Materials Research
4 Probes and Tools for Biomolecular Materials Research
Pages 76-115
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From page 76... ...
A further motivation for these studies is the expectation that the process of learning how work is performed at the very small subcellular scales should pave the way for the development of functional biomolecular materials in future nanotechnological applications. Biological systems consist of collections of interacting molecules (proteins, carbohydrates, lipids, and nucleic acids)
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From page 77... ...
The techniques should also apply equally well to the broader class of man-made biomolecular materials. The new experimental probes of biomolecular materials are expected to have a major impact on future science and technology, including imaging methods based on novel optical and electron microscopic techniques, new synchrotrons, and X-ray free electron lasers, which provide ultrashort and extremely intense pulses.
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From page 78... ...
spectroscopy or macromolecular crystallography, which are used primarily for elucidating the structure of single protein molecules at angstrom resolution, three-dimensional cryo-electron microscopy (cryo-EM) is emerging as a powerful tool for capturing images, albeit at lower resolution, of directed- or self-assembled collections of biological molecules, complexes, and machines that function in concert.
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From page 79... ...
distribution of supramolecular assemblies of biological complexes, molecular machines, and motors. Figure 4.2 shows images of the actin cytoskeleton in the slime mold Dictyostelium discoideum using state-of-the-art cryo-ET.
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From page 80... ...
The majority of CTs and MRIs currently in use are able to visualize objects about 1 cubic millimeter in size, a useful scale for clinical diagnosis. By the same token, cryo-ET and single particle cryo-EM should allow researchers to visualize cells, their organelles, and the crucial biomolecular assemblies at a near molecular resolution, the appropriate spatial dimension to uncover their functional behavior.
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From page 81... ...
A big disadvantage with which optical microscopy normally contends is its limited spatial resolution relative to molecular dimensions. The spatial resolution for discriminating two objects in a conventional optical microscope is limited to about 250 nm owing to diffraction of the light by the imaging objective.
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From page 82... ...
Certain fluorescent proteins, organic dyes, and pairs of closely spaced cyanine dyes are photoswitchable between fluorescent and nonfluorescent metastable states using two different wavelengths. Again, switching these probes off is saturable, allowing the equivalent of STED narrowing of the PSF but at much lower laser intensities.
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From page 83... ...
The second approach does not require an X-ray lens but requires a source with a high brilliance, such as a third-generation synchrotron source or the upcoming fourth-generation free-electron X-ray laser source. Newly developed X-ray imaging techniques are expected to have a major impact on biomolecular materials research by facilitating fundamentally new ways of characterizing events at the nanometer scale and also as a function of time.
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From page 84... ...
FIGURE 4.4 (Left) Schematic of a zone-plate-based full-field three-dimensional X-ray micro scope operating in phase contrast mode.
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From page 85... ...
It has been shown that when the no-density region is larger than the electron density region, the phase information is uniquely encoded in the diffraction pattern and can be recovered directly by an iterative process that takes advantage of the knowledge that the electron density outside the object is zero and within the object is positive. The first successful experimental demonstration of coherent diffraction imaging was carried out in 1999.
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From page 86... ...
X-ray free electron lasers also offer promising prospects with their ultrashort and extremely intense pulses. In this case, radiation damage could be circumvented by recording the diffraction pattern from single macromolecules before they are destroyed.
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From page 87... ...
Several recent studies of higher-order assembly of cytoskeletal microtubules and filamentous actin have in fact demonstrated the importance of the combined techniques in elucidating such partially ordered hierarchical structures. Neutron Scattering Neutron scattering and diffraction provide detailed information on the structure and dynamics of biomaterials and systems across time and length scales that range from pico- to nanosecond of time resolution and from 1 to 10,000 Å of spatial resolution.
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From page 88... ...
This is beneficial for determining enzyme mechanisms, for studies of ligand binding interactions and, since complete D2O water molecules are prominent in neutron density maps, for detailed analysis of the structure and dynamics of water in hydra tion layers at the protein-solvent interface. Neutron diffraction can determine the pattern and extent of H/D isotope substitution in proteins, the solvent accessibility of individual amino acids, the mobility and flexibility of interesting domains, and the H/D exchange dynamics themselves.
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From page 89... ...
At the mesoscale, neutron scattering is sensitive to the bulk hydrogen atom content and composition of materials and can be used to characterize and determine the structure of mixed and complex systems and phases. For example, the bulk neutron scattering characteristics of proteins, nucleic acids, lipids, and carbohydrates all differ significantly from one another (Figure 4.7)
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From page 90... ...
So-called single-molecule biophysics reveals the mechanics, biochemistry, structural biology, and dynamics of biomolecular processes, complementing classical methods for understanding them. Some important aspects of biomolecular function are usually obscured in studies of ensembles of molecules due to the averaging of heterogeneities and dynamic variations among their individual functional units.
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From page 91... ...
Dramatic progress in understanding biological macromolecules, materials, and cellular function has derived from genetics, structural biology, and biochemical experiments on ensembles, solutions, and suspensions of proteins and nucleic acids and in live cells. High-resolution structures derived from X-ray crystallography and cryo-EM are revealing snapshots of particular states in the reaction pathways.
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From page 92... ...
The primary output of molecular motors and DNA p rocessing enzymes is progress along cytoskeletal or nucleic acid tracks. The indi vidual motions are difficult to synchronize in an ensemble, so only the average progress is detected.
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From page 93... ...
Total Internal Reflection Fluorescence Microscopy Detecting the fluorescence from single organic dyes (for example, rhodamine) , fluorescent peptides (for example, green fluorescent protein)
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From page 94... ...
(A) Two tightly focused infrared optical beams are depicted as pink waists.
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From page 95... ...
The excitation volume is often reduced by using the tightly focused exciting beam in a confocal microscope or the evanescent electromagnetic field that is present near a total internally reflective interface, giving total internal reflection. Figure 4.9a shows this geometry for a single-molecule total internal reflection fluorescence (TIRF)
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From page 96... ...
(B) Position of a rhodamine fluorescent probe attached to a myosin V molecular motor while the motor translocates along a filament of the cytoskeletal protein, actin.
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From page 97... ...
Typical molecular simulations are based on molecular dynamics, Monte Carlo methods, or Brownian dynamics methods. There are many examples of problems pertinent to biomolecular processes and biomaterials that could be addressed using these methods.
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From page 98... ...
The development of multiscale simulations that can accurately describe bio molecular assemblies beginning from their fundamental protein building blocks represents a new challenge in terms of both their underlying theoretical basis and their computational implementation. As one illustrative example, Figure 4.11 depicts the multiple scales that exist for the actin filament (the major component of the cellular cytoskeleton)
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From page 99... ...
However, these methods are extremely computation-intensive and difficult to apply as the complexity of the phenomena being studied increases. The understanding of biomolecular processes and concomitant design of biomolecular materials will be greatly aided by faster and more efficient algorithms for the simulation of spatially resolved, stochastic dynamic phenomena in cells.
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From page 100... ...
It is possible that developing such methods will also impact computational investigation of phenomena that occur on larger scales (for example, collections of cells or macroscopic biomaterials with built-in nanoscale functionality)
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From page 101... ...
Access to High-Performance Computing Environments Supercomputer centers play an important role in computational research on bimolecular materials and processes. However, many computations for bio materials design require faster swaps of information than can be provided by grid computing or require resources that are more dedicated than are available at current supercomputer centers.
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From page 102... ...
Computational studies allow elaborating the consequences of mechanistic hypotheses and the calculation of material properties for specific systems. While synergy between computation and experimentation can provide some insights that go beyond the specific system studied, the development of overarching principles that govern the behavior of classes of systems requires theoretical studies as well.
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From page 103... ...
problems will be a very important component of the arsenal of tools for understanding biomolecular processes and for developing biomaterials with precise functional properties.
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From page 104... ...
Proteins, too, are created by the differential arrangement of only 22 amino acids linked together through identical amide bonds. Nature uses this simplistic monomer set to create an infinite and complex library of proteins with functions ranging from cellular signaling and transport to complex molecule synthesis, assembly, and degradation.
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From page 105... ...
Ethylene and functionalized vinyl monomers are polymerized around a template and this analyte molecule can then be removed, which endows such systems with molecular recognition properties. Existing materials have also been commonly utilized and modified for drug and nucleic acid delivery.
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From page 106... ...
Incredible opportunities exist in developing unique catalytic systems that promote coupling in a user-friendly and efficient manner and can be carried out in the presence of oxygen and water to create highly functional materials.
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From page 107... ...
By hijacking and engineering the promiscuous cellular machinery, researchers have also shown that novel materials can be created by incorporating nonnatural amino acids into protein structures containing functional groups not normally found in biological systems. This allows the specific placement of reactive sites in a polypeptide such as alkynes and azides that can be utilized for specific and selective bioconjugation with, for example, the click reaction.
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From page 108... ...
For example, if synthetic viruses can be built to deliver nucleic acids into cells in a specific, efficient, and nontoxic manner, a paradigm shift from small molecule to macromolecular therapeutics could occur and revolutionize modern medicine. In addition, multivalent materials have been shown to serve as inhibitors and effectors for various biochemical pathways and have promise as novel drugs and research tools.
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From page 109... ...
Novel ligation chemistries with nucleosides and amino acids have also created completely new materials such as peptoids and peptide nucleic acids, which are not subject to enzymatic degradation in biological systems and may yield diverse applications from artificial transcription factors to antisense agents. The challenges with exploring and exploiting such materials lie in the fact that researchers are limited by the lack of biological knowledge and the unavailability of functional peptides and other biomolecules to enhance biomaterial performance.
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From page 110... ...
Novel supramolecular polymers have been created by peptide amphiphiles (Figure 4.16)
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From page 111... ...
Many other novel methods of monodiperse nanoparticle formation are beginning to unfold using novel molding and templating techniques. Tremendous opportunities exist in examining noncovalent polymerization and assembly methods
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From page 112... ...
that yield biomolecular structures. Although challenges remain in understanding and controlling the creation, morphology, and reproducibility of such structures, exceptional properties will result once researchers understand and can manipulate these processes.
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From page 113... ...
• Challenge: Achieving angstrom resolution in electron and X-ray imaging -- Opportunity: Unprecedented elucidation, at the molecular level, of the structural and operational principles of many important cellular processes • Challenge: Studying the structure, dynamics, and kinetics of assemblies of biomolecular systems using X-ray scattering techniques -- Opportunity: Elucidating the dynamical processes involved in RNA genomes packing into viral capsids and probing the collective behavior of motors moving on their natural tracks • Challenge: Probing reactions using neutron scattering at timescales of microseconds or longer -- Opportunity: The ability to probe dynamical behavior as well as struc tural correlations in biomolecular materials and processes • Challenge: Developing techniques for seeing correlations in space and time (for example, neutron correlation spectroscopy and correlated neutron imaging) rather than in q (momentum)
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From page 114... ...
-- Opportunity: Unprecedented control over the synthesis of new and complex biomolecular materials
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From page 115... ...
Bacon, G.E., Neutron Diffraction, Glasgow, New York: Oxford University Press, 1975. Grunewald, K., O
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