Native protein structures applications

This chapter describes the self-assembly of non-native protein fibers known as amyloid fibrils and the development of these fibrils for potential applications in nanotechnology and biomedicine. It extends an earlier review by the author on a related topic (Gras, 2007). In Section 1, the self-assembly of polypeptides into amyloid fibrils and efforts to control assembly and any subsequent disassembly are discussed. In Section 2, this review focuses on the important role of surfaces and interfaces during and after polypeptide assembly. It examines how different surfaces can influence fibril assembly, how surfaces can be used to direct self-assembly in order to create highly ordered structures, and how different techniques can be used to create aligned and patterned materials on surfaces following self-assembly. 

One of the primary mechanisms of protein degradation is the loss of globular structure [118, 119]. This process, termed denaturation, leads to a partially or completely unfolded species which usually lacks any of the biological activity of the native protein. A variety of methods have been employed to monitor the denaturation of proteins, including fluorescence, infrared, nuclear magnetic resonance (NMR), and CD spectroscopy. As CD is very sensitive to changes in both secondary and tertiary structure, its application to the study of protein folding... 

A good example of application is given by the protein structural changes of bovine ribonuclease A in the course of its denaturation by pressure. The UV spectrum of RNase is dominated by the absorbance of tyrosine - this RNase does not contain tryptophan. As shown in Figure 6, an increase of pressure from 1 to 500 MPa results in a blue-shift of the 4th derivative maximum from 285.7 0.05 to 283.5 0.05 nm. This shift of 2.2 nm corresponds to an increase of the mean dielectric constant from 25 to 59. It is characteristic of the exposure to the aqueous solvent of part of the 6 tyrosines, as it is expected for a partly denaturation. The transition is fully reversible with clear isosbestic points. The pressure effect can therefore be described by a simple two-state model between the native (e,. = 25) and the partially denatured (e,. = 59) state. A simulation on the basis of this model permitted us to determine the thermodynamic parameters of this transition AG° = 10.3 kJ/mol and AV = - 52 ml/mol. A comparison with results obtained by other methods indicates that the (e,. = 59) state corresponds to an intermediate in the defolding process which has molten globule like characteristics [12]. It thus appears that fourth derivative... 

Ferritin induced nanoparticle synthesis was adapted from a number of different synthetic strategies reliant upon the physical nature of ferritin. For instance, ferritin can readily exist in two stable forms (native ferritin with an intact iron oxide core or apo-ferritin lacking a mineral core) owing to the enhanced structural integrity of the protein shell. As a result, two general reaction schemes were adopted. The first route utilized the iron oxide core of native or reconstituted ferritin as a precursor to different mineral phases and types of iron nanoparticles, while the second invokes mineralization within the empty cavity of apo-ferritin. In the latter approach, the native protein must be demetallated by reductive dissolution with thioglycolic acid to yield apo-ferritin. Ultimately, apo-ferritin provides a widely applicable means to the synthesis of various nanoparticle compositions under many conditions. 

Biomolecular NMR spectroscopy is applicable to both liquid-and solid-state samples. Liquid-state NMR spectroscopy, in which molecules are dissolved in a variety of different solvents and studied at ambient temperatures, is a powerful tool to derive information on the stmcture of proteins and nucleic acids, as well as their complexes with each other and small molecules, ions, and solvents. Liquid-state NMR can be applied not only to native folded states of proteins, but also to intrinsically unstmctured proteins as well as proteins in their unfolded state and under nonphysiological conditions (i.e., in organic solvents). Figure 1 provides an overview on the number of protein structures determined by liquid-state NMR spectroscopy. 

This approximate law holds for both native structure [22] and folding dynamics [2], In this regard, this wrapping motif may be regarded as a structural element that captures the basic component of energy transduction from hydrophobic association to structure formation. Furthermore, it implies that a fundamental constraint in protein architecture applicable to native structures applies also throughout the folding trajectory. 

The denaturation of protein involves loss of their tertiary and secondary structures. This typically occurs by application of some external stresses, out of which thermal, interfacial and dehydration-related stresses are the most important stresses causing denaturation of proteins in drying processes. These stresses disrupt the tertiary structure, and subsequently the a-helix and j3-sheets of native proteins are turned into unfolded random shapes. When a protein is unfolded, the hydrophobically buried sites are exposed to the solvent and subsequently interact with interfaces and other unfolded polypeptides. The unfolded protein allows subsequent cross-linking interactions such as protein-protein hydrophobic, electrostatic, and disulfide-sulfhydryl interactions. These interactions result in aggregation, coagulation, and, finally, precipitation (Pelegrine and Gasparetto, 2005 Anandharamakrishnan et al, 2007). 

A frequently used approach to study the thermal stability of proteins is to incubate a protein solution at an elevated constant temperature and to observe the change of certain physical parameters (e.g., CD, IR absorbance, enzyme activity) over periods of minutes or hours. Such measurements deliver precious information for the practical application of the protein in question. On the other hand, it is impossible to extract thermodynamic or structural parameters firom such measurements, as they reflect the loss of native protein caused by a variety of processes. Irreversible thermal denaturation involves complex mechanisms and can lead to precipitation. The rates of such reactions depend on the concentration the rate constants depend on temperature and solution conditions. The order of such reactions can vary from 1 to FTIR has the advantage that it at least allows clear identification of -aggregation in the changes in the amide I band (1600-1700 cm ) of the infrared spectrum. The band component at around 1618 cm reliably reflects the progress of P-aggregation. ... 

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