Polypeptide denaturation

After semi-preparative RP-HPLC, homogeneous fractions are pooled, lyophilized, and redissolved in an aqueous solvent. In the case of sparingly soluble polypeptides, denaturing solutions (e.g. 6 M guanidinium-HQ) can be used which do not affect the subsequent reaction of probe removal. This is carried out by the addition of an organic base (e.g. NEta, see Protocol 4) which catalyses the p-elimination typical for the Fmoc protection giving the corresponding dibenzofulvene derivative and free polypeptide Scheme 7), A final semi-preparative RP-HPLC separates the latter from the probe by-product. 

In the case of poorly soluble polypeptides, denaturing solutions such as 6 M guanidinlum HCI can be used. 

Polypeptide denaturation is often partial under these conditions A complete denaturation may be obtained in 6 M guanidium choride, and further reduction with 5 mM DTT. This treatment yields a polypeptide fully accessible to endoproteases. 

Figure 6.1 A polypeptide chain is extended and flexible in the unfolded, denatured state whereas it is globular and compact in the folded, native state.

To answer the question whether the ds-transisomerization of the bridged polypeptides with a Ala-Gly-Pro sequence represents the rate-determining step, the following experiment was carried out The polypeptide with a chain length n = 8 was denaturated in a rapid reaction with a temperature jump from 9.2 to 30 °C and subjected to renatura-tion at 9.2 °C after an incubation time of 25 s. In a second and a third experiment, the incubation in the coiled state was prolonged respectively to 75 and 125 s. It could be observed that the amplitude of the rapid phase depends on the time that lapses between the denaturation and renaturation (Fig. 32). 

Baler, R., Welch, W.J., Voellmy, R. (1992). Heat shock gene regulation by nascent polypeptides and denatured proteins hsp70 as a potential autoregulatory factor. J. Cell. Biol. 117, 1151-1159. 

Both the heat and cold shock response are universal and have been studied extensively. The major heat shock proteins (HSPs) are highly conserved. They are involved in the homeostatic adaptation of cells to harsh environmental conditions. Some act as molecular chaperones for protein folding, while others are involved in the processing of denatured polypeptides whose accumulation would be deleterious. The cold shock results in the transient induction of cold shock proteins (CSPs), which include a family of small acidic proteins carrying the cold shock domain. The CSPs appear to be involved in various cellular functions such as transcription, translation and DNA recombination. 

First, we investigated whether Sh I could be reduced under denaturing conditions, exposed to the hydrogen fluoride cleavage procedure we intended to use, then reoxidized and refolded successfully. The toxicity of the resulting polypeptide (50% yield) was the same as that of the untreated natural toxin. 

When collagen is heated, it is denaturated, losing its structure the polypeptide chains separate and unwind, and, as the collagen cools down, it soaks up surrounding water and forms gelatin. 

The changes in structure of denatured nuclease as a function of urea concentration (Fig. 3) suggest that, as hydrophobic interactions are weakened and the backbone becomes more highly solvated, the chain expands gradually. The data presented by Millet et al. in this volume suggest that this expansion does not continue asymptotically as predicted by simple polymer physical chemistry. This is the behavior expected for a polypeptide chain trapped in a small region of conformation space. Most, perhaps all, of the conformations accessible in the expanded denatured state may have a native-like topology. 

Although additional experiments and simulations are needed to determine how much of reality is captured in this model, it does explain one important property of proteins their rapid rate of refolding, which is independent of denaturation conditions. If the polypeptide chain is unable to escape from this steric trap and access conformations with the wrong topology, it could never wander far from the folded conformation and thereby avoid incorrect side chain/side chain interactions. 

The application of ROA to studies of unfolded and partially folded proteins has been especially fruitful. As well as providing new information on the structure of disordered polypeptide and protein sequences, ROA has provided new insight into the complexity of order in denatured proteins and the structure and behavior of proteins involved in misfold-ing diseases. All the ROA data shown in this chapter have been measured in our Glasgow laboratory because, at the time of writing, ROA data on typical large biomolecules had not been published by any other group. We hope that this review will encourage more widespread use of ROA in protein science. 

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. 

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