Renaturation transitions

Calorimetric studies have been made on proteins S4, S7, S8, S15, S16, S18, Lll, and L7 (Khechinashvili et al., 1978 Gudkov and Behike, 1978). Most of these proteins displayed a cooperative tertiary structure in solution. Proteins S4, S7, SI5, and SI8 were extracted from the ribosome by a urea-LiCl technique followed by renaturation, whereas proteins S8, S16, and Lll were prepared by the mild isolation method. A calorimetric study on protein SI showed a noncooperative transition around 70-80 C, suggesting a flexible tertiary structure (L. Giri, unpublished). 

The study of temperature effects on the reactivation of an enzyme that has been completely unfolded allows one to distinguish between reactivation (referring to kinetic analysis exclusively) and renaturation, the latter of which would reflect both the refolding transition and the formation of misfolded or aggregated byproducts. 

Some results. Rapid kinetic methods have revealed that enzymes often combine with substrates extremely quickly,60 with values of k] in Eq. 9-14 falling in the range of 106 to 108 M 1 s . Helix-coil transitions of polypeptides have relaxation times of about 10-8 s, but renaturation of a denatured protein may be much slower. The first detectable structural change in the vitamin A-based chromophore of the light-operated proton pump bacteriorhodopsin occurs in - 5 x 10 8 s, while a proton is pumped through the membrane in... 

Renaturation. The effects of i) reduction of the disulfide bond of p-lactamase, ii) renaturation buffer pH, iii) concentration of protein, iv) GuHCl in the denaturing solution and finally v) sucrose concentration on the reversibility of the unfolding transition were investigated. 

It is important to mimic not only the static structures but also their dynamic properties. Conformational transitions, changes of folds, denaturation, and renaturation of biopolymers can be understood better if lattice dynamics, phase transitions, amor-phization of crystalline amino acids and small peptides are studied and compared with those in synthetic polyaminoacids and in two-dimensional layers at the interfaces. Variable-temperature [44, 64-84] and variable-pressure [29, 81, 82, 85-134] IR- and Raman spectroscopy, inelastic neutron scattering, SAXS, NMR, X-ray and neutron diffraction, DSC are applied to study the structure and dynamics of crystalline amino acids, small peptides, synthetic polymers, interface layers and biopolymers [73-153]. 

Denaturation and Renaturation are Sharp Cooperative Transitions, with Latent Heat... 

Figure 6 Example of detection of mutations in form of mismatches in ds-DNA by temperature gradient electrophoresis. (A) Denaturation-renaturation cycle, with a schematic representation of the mechanism demonstrating that a base-pair exchange ( - ) is transferred into a mismatch >, < ). (B) Schematic drawing of the transition curves, showing that the denaturation temperature of the segment, which carries the mutation is lowered significantly. W, wild-type sequence, (-I-) strand w, wild-type sequence, (-) strand M, mutated sequence, + ) strand m, mutated sequence, -) strand , point mutation. (Reproduced with permission from Riesner D, Henco K, and Steger G (1991) Advances in Electrophoresis, vol. 4, pp. 169-250 VCH.)...

The gelatin-type of triple heUx may be considered a type of crystal indeed, the denaturing and renaturing of these gels is a first-order transition. However, it has been modeled as a nucleated, one-dimensional crystallization, as opposed to the ordinary three-dimensional crystallization of bulk materials. 

Denaturation and Renaturation When DNA molecules are heated to certain temperature (e.g., lOO C), the two polynucleotide strands separate. The transition from the double strand (original form) to the single strand (denatured form) can be observed by the change in optical density at 260 nm. The plot of the optical density versus temperature gives a sinusoidal curve that is similar to an acid-base titration curve. In Figure 17.8 the point Tin, which corresponds to the equivalence point in an acid-base titration, is the hypochromic point and denotes where a mixture of the native and denatured strands occurs. 

A useful and important parameter, readily obtained from these measurements, is the helix-to-coil transition temperature, the temperature at which half of the helical structure is lost or altered. The transition, also called melting or denaturation, is highly cooperative and is also pH dependent. Indeed, the transition occurs within 0.1 pH unit instead of several units as might be expected from a consideration of the titration values of the individual bases. Below the transition temperature separated complementary strands automatically reassociate (renaturation). 

Hypochromism With respect to DNA, a reduction in the absorbance of ultraviolet light of wavelength of about 260 nm that accompanies the transition from random-coil denatured strands to a double-strand helix. It can be used to track the process of Denaturation or renaturation. 

The phenomenon of denaturation and renaturation can ideally be treated using a two-state model involving the transition between ordered double helices and disordered or random coils. When the transition monitored by A260, fot... 

Fig. 5.27. Transition curve of elastase denatured by GuHCI at pH 5.4 (from Ghelis and Zilber, 1982) denaturation process ( , ) renaturation (0,1 V).

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