Denaturation random coil

Chelation and site-directed mutagenesis studies indicate the zinc is essential for formation of an enzymatically active form of the enzyme. The zinc-depleted enzyme shows mostly unchanged secondary structure as determined by CD spectroscopy, but not a fully denatured random coil as obtained by the presence of guanidinium hydrochloride. The zinc may therefore be stabilizing a local conformation of the protein that is critical for activity. 

The shapes of solutes are also important in their retention behavior it has been shown that DNA restriction fragments (rod-shaped) have Km values that are more sensitive to molecular weight than those obtained with denatured (random-coil) proteins. In fact, the SEC parameter governing the retention is the hydrodynamic volume of the solute, which is related to its radius of gyration, Rg. The molecular weight of a solute is related to its radius of gyration by Eq. 14.12 ... 

Thermodynamically, the stability of the three-dimensional fold of an enzyme can be described as the equilibrium between the folded native state (N) and the denatured random coil configuration (D). The temperature function of the free... 

N(r) can be related to the fractal dimension D of the protein-surfactant complex by the equation N r) = (r/R jP. For a freely diffusing micelle the fractal dimension would be 3, but in a complex the distribution of micelles is dictated by the topology of the polypeptide backbone, so that the fractal dimension is less than 3. At 1% by weight of lithium n-dodecylsulphate, D is 2.3 and decreases to 1.76 at 3%, consistent with a transition from a compact state to a more open random coil in which a string of constant-sized micelles are distributed along the hydrophobic patches of a denatured random coil, although the coil will be restrained by the 17 disulphide bonds in the BSA structure. 

Changes in conformation of the protein molecule have been implicated in each of these separation problems (see review of Ref. 33). It is well known that proteins commonly exist in either native (folded) or denatured (random coil) conformations. This offers a simple explanation for the appearance of two separate bands following the injection of a single (pure) protein. Figure 10 illustrates an example of this kind. Here the enzyme papain was separated by reversed-phase gradient elution using different conditions of mobile phase pH and temperature (as noted in Fig. 10a). Higher temperatures and/or lower pH favor the denatured protein, which is observed to elute at about 22 min. The native protein elutes at about 11 min. 

Indirect determination of the enthalpy of unfolding assumes the knowledge of the equilibrium as a function of temperature. Starting from spectroscopic data spectroscopic signal for 100% denaturated (random coil) sample and 100% native protein was determined. The temperature range where protein transitions from native to denatured form was covered. Fraction of native protein as a function of temperature and the fraction of unfolded protein as a function of temperature /n and fo respectively, were defined in terms of measured absorbance A(T) as ... 

For instance, one would like to know the types of structures actually present in the native and denatured proteins.. .. The denatured protein in a good solvent such as urea is probably somewhat like a randomly coiled polymer, though the large optical rotation of denatured proteins in urea indicates that much local rigidity must be present in the chain (pg. 4). 

First, proteins refold from the denatured state, not from the hypothetical random coil state. It is the starting point of all refolding reactions, whether in a cell or in a test tube. To understand any chemical reaction, structural features of the reactant and the product must be compared to quantify the changes that occur, for these changes define the reaction. 

Fig. 6. Spectral monitoring of the thermal denaturation of the highly helical, Ala-rich peptide Ac-(AAAAK)3AAAA-YNH2 in D20 from 5 to 60°C, as followed by changes in the amide V IR (left) and VCD (right). IR show a clear shift to higher wavenumber from the dominant a-helical peak (here at an unusually low value, 1637 cm-1, due to full solvation of the helix) to a typical random coil value ( 1645 cm-1). VCD loses the (—,+,—) low-temperature helical pattern to yield a broad negative couplet, characteristic of a disordered coil, at high temperature. Spectra were normalized to A = 1.0 by 45°C.

Thermally denatured proteins have been studied for a variety of systems using FTIR and VCD. The resulting high-temperature spectra often reflect the characteristics seen earlier for random coil peptides as well as that seen for the unstructured casein. Particularly the amide I IR bands show a frequency shift to center on a broadened band at 1645-50 cm-1. The amide I VCD loses its distinctive character (Fig. 11) and tends toward... 

Fig. 11. Amide F thermal denaturation spectra for ribonuclease A as followed by FTIR (left) and VCD (right), which show the IR peak shifting from the dominant /3-sheet frequency (skewed with a maximum at 1635 cm-1) to the random coil frequency ( 1645-1650 cm-1) and the VCD shape changing from the W-pattern characteristic of an a + p structure to a broadened negative couplet typical of a more disordered coil form. The process clearly indicates loss of one form and gain of another while encompassing recognition of an intermediate form. (This is seen here most easily as the decay and growth back of the 1630 cm-1 VCD feature, but is more obvious after factor analysis of the data set, Fig. 15).

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

The CD spectra of nine proteins in 6 M Gdm-HCl were studied by Cortijo etal. (1973). Those proteins with disulfide bridges were reduced and carboxymethylated. The spectra of individual proteins were not reported, but the range of values at wavelengths from 240 to 210 nm was given. The [0]222 values ranged from —800 to —2400 deg cm2/dmol. From this substantial variation, Cortijo etal. (1973) concluded that the proteins studied are not true random coils in 6 M Gdm-HCl, because random coils should have CD spectra essentially independent of amino acid composition and sequence. The observed variation was attributed to differences in the conformational distribution between allowed regions of the Ramachandran map or to residual interactions between different parts of the chain that are resistant to Gdm-HCl denaturation. 

IV. Reconciling the Random Coil with a Structured Denatured State. 257... 

An excluded-volume random-coil conformation will be achieved when the solvent quality exceeds the theta point, the temperature or denatu-rant concentration at which the solvent-monomer interactions exactly balance the monomer—monomer interactions that cause the polymer to collapse into a globule under more benign solvent conditions. A number of lines of small-angle scattering—based evidence are consistent with the suggestion that typical chemical or thermal denaturation conditions are good solvents (i.e., are beyond the theta point) and thus that chemically or thermally unfolded proteins adopt a near random-coil conformation. 

The geometric properties of highly denatured states appear to be consistent with those expected for a random-coil polymer. For example, proteins unfolded at high temperatures or in high concentrations of denaturant invariably produce Kratky scattering profiles exhibiting the monotonic increase indicative of an expanded, coil-like conformation (Fig. 1) (Hagihara et al., 1998 see also Doniach et al., 1995). Consistent... 

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