Comparison with native proteins

Limited digestion of globular soy proteins with rennin affords a modified protein preparation which retains a high molecular weight (47). Whipping quality, measured by foam volume and stability, was superior in comparison with native proteins. The limited rennin proteolysis of soy was identified as a key factor in functionality, since this modification conferred improved solubility. 

This section indicates that is possible to transfect cells with GFP fusion proteins of the AR families. The co-localization of these proteins with fluorescent ligands provides a link that allows comparison with native cell systems that do not possess such useful tags but that bind the fluorescent ligands. However, an additional utility is provided by the ability to label receptors in native systems with GFP, which provides the opportunity for receptor translocation studies in native cells. This has been accomplished on two levels. First, by in vitro transfection of native tissues or cells (see Fig. 1C) and second by creating transgenic mice harboring the GFP-AR constructs (see Chapter 7). 

Y. Mely and D. Gerard, Structural and ion-binding properties of an SlOOb protein mixed disulfide Comparison with the reappraised native SlOOb protein properties, Arch. Biochem. Biophys. 279, 174-182 (1990). 

Peptide Vaccines Peptide vaccines are chemically synthesized and normally consist of 8-24 amino acids. In comparison with protein molecules, peptide vaccines are relatively small. They are also known as peptidomimetic vaccines, as they mimic the epitopes. Complex structures of cyclic components, branched chains, or other configurations can be built into the peptide chain. In this way, they possess conformations similar to the epitopes and can be recognized by immune cells. An in silico vaccine design approach has been used to find potential epitopes. A critical aspect of peptide vaccines is to produce 3D structures similar to the native epitopes of the pathogen. 

When the urea and thiol are removed by dialysis (see p. 78), secondary and tertiary structures develop again spontaneously. The cysteine residues thus return to a suf ciently close spatial vicinity that disulfide bonds can once again form under the oxidative effect of atmospheric oxygen. The active center also reestablishes itself In comparison with the denatured protein, the native form is astonishingly compact, at 4.5 2.5 nm. In this state, the apolar side chains (yellow) predominate in the interior of the protein, while the polar residues are mainly found on the surface. This distribution is due to the hydrophobic effect (see p. 28), and it makes a vital contribution to the stability of the native conformation (B). 

Reverse micellar extraction (RME) is another attractive LLE method for DSP of biological products, as many biochemicals including amino acids, proteins, enzymes, and nucleic acids can be solubilized within and recovered from such solutions without loss of native function/activity. In addition, these systems offer low interfacial tension, ease of scale-up, and continuous operation. RME offers a number of unique, desirable features in comparison with ATPE, which has been extensively studied ... 

Contemporary analytical methods (see Campbell, 1996) permit the ligand binding sites and the conformational changes they produce on the native protein to be identified. Even with proteins which have not been isolated but with amino acid sequences predicted from DNA studies, searching data banks allows comparisons with proteins whose structure and function are already known. 

The molar refractions of the amino acids were determined by measurements on their aqueous solutions and the expanded Lorenz-Lorentz equation. The refractive indices of a number of proteins were calculated from their amino acid compositions and the values for the refraction of the amino acid residues. These calculated results are in good agreement with those experimentally determined, demonstrating that refractive index is a unique characteristic of a protein. A comparison of the refractive index of heat denatured /3-lactoglobulin with the native protein demonstrated that changes in structure produced a small change in refractive index, not associated with a change in volume. 

Proteins that possess a quaternary structure are composed of several separate polypeptide chains held together by noncovalent interactions. When such proteins are examined under dissociating conditions (e.g., 8 M urea to weaken hydrogen bonds and hydrophobic interaction, 1 m/lf mercaptoethanol to disrupt disulfide bonds), the molecular weight of the component polypeptide chains can be determined. By comparison with the native molecular weight, it is often possible to determine how many polypeptide chains are involved in the native structure. 

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... 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

Stiasny et al, 1996). This region fell outside the proteolytically released ectodomain of the TBEV E protein and SFV El structures, and its conformation is not known in the native state. Although the trimeric form of an alphavirus El protein has been analyzed by proteolysis (Gibbons and Kielian, 2002), no high-resolution structures of the low-pH trimerized state are yet available for alphaviruses or flaviviruses, so a further structural comparison with flu cannot be made at this time. 

As stated earlier, proteins can be denatured by heat or by chemical denaturants such as urea or guanidium chloride. For many proteins, a comparison of the degree of unfolding as the concentration of denaturant increases has revealed a relatively sharp transition from the folded, or native, form to the unfolded, or denatured, form, suggesting that only these two conformational states are present to any significant extent (Figure 3.56). A similar sharp transition is observed if one starts with unfolded proteins and removes the denaturants, allowing the proteins to fold. 

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