Force-induced denaturation of RNA

We quantitatively describe an RNA molecule under the influence of an external force exerted at its two ends as in a typical single-molecule experiment. Our calculation incorporates the interactions between nucleotides by using the experimentally determined free energy rules for RNA secondary structure and models the polymeric properties of the exterior single-stranded regions explicitly as elastic freely jointed chains. We find that despite complicated secondary structures, force-extension curves are typically smooth in quasi-equilibrium. We identify and characterize two sequence/structure-dependent mechanisms that, in addition to the sequence-independent entropic elasticity of the exterior single-stranded regions, are responsible for the smoothness. These involve compensation between different structural elements on which the external force acts simultaneously and contribution of suboptimal structures, respectively. We estimate how many features a force-extension curve recorded in nonequilibrium, where the pulling proceeds faster than rearrangements in the secondary structure of the molecule, could show in principle. Our software is available to the public through an "RNA-pulling server."     

The denaturation of trypsin

The denaturation of α- and β-trypsin in alkaline and neutral solution was studied. The denaturation of α-trypsin was a strict second-order reaction at neutrality. However, the denaturation of β-trypsin was not a pure secondorder reaction at the same pH. Calcium ion retarded the rate of β-trypsin denaturation to a greater extent than that of α-trypsin. In alkaline solution, trypsin has very short half life ($\text{t}{}_{\mathrm{<mtext>case1</mtext><mtext>2</mtext>}}\text{< 30 minutes}$). On the other hand, the denaturation of immobilized trypsin in alkaline solution is a first-order reaction as is immobilized chymotrypsin. The rates of these two denaturations are similar. Calcium ion does not affect the rate of trypsin denaturation in alkaline solution.     

Cold denaturation of proteins

This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly temperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reevaluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive contributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed.     

Cold denaturation of myoglobin

The stability of the structure of sperm whale metmyoglobin has been studied in various solutions, in the temperature range -8 degrees C to 100 degrees C, by scanning microcalorimetry, light absorption, circular dichroism, nuclear magnetic resonance spectroscopy and viscosimetry. It has been shown that in 10 mM-sodium acetate solutions (pH 3.5 to 3.9) the protein molecule undergoes a reversible conformational transition into a non-compact disordered state not only when the solution is heated above room temperature but also when it is cooled. In this state the protein does not have a tertiary structure, although it retains some residual ellipticity, which may be caused by the fluctuating alpha-helical conformation of the unfolded polypeptide chain. The disruption of the native protein structure both on cooling (cold-denaturation) and on heating (heat-denaturation) proceeds in an "all-or-none" manner, with a significant and similar increase of the protein heat capacity, but with inverse enthalpic and entropic effects: the enthalpy and entropy of the protein molecule decrease during cold-denaturation and increase during heat-denaturation.     

High-pressure denaturation of apomyoglobin

The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.     

The denaturation of orosomucoid

Highly purified human orosomucoid exhibits apparent multistate behavior on thermal denaturation, with ΔH(cal) for the transition being 119 kcal/mol at pH 7.4. Asialoorosomucoid denatured similarly. A domain structure could not be demonstrated for orosomucoid with cyanogen bromide fragments, although some reannealing of these did occur. It is suggested that the source of the apparent multistate behavior may lie in the existence of polypeptide variants, known to be present in orosomucoid. Although some polymerization occurs on heating, the unpolymerized material shows reversible thermal denaturation behavior. The presence of low concentrations of ethanol induces a significant endotherm which moves to lower temperatures with increasing ethanol concentration. This endotherm was irreversible in the continued presence of ethanol.     

Cold denaturation of alpha-lactalbumin

We have investigated the thermal unfolding of bovine alpha-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native alpha-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride-induced unfolding of alpha-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride-induced unfolding transition at 261 K. Our results indicate that the molten globule state of alpha-lactalbumin undergoes cold denaturation as the native alpha-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride-induced unfolding behavior of alpha-lactalbumin.     

Heat denaturation of ribonuclease

One of the main and, chronologically, perhaps one of the first questions in the study of globular protein heat denaturation is that of the applicability of the “all or none” principle to this process, i.e., whether the transition of globular protein from the native into the denaturated state occurs abruptly, without intermediate, thermodynamically stable forms or there are several successive transitions. Despite an intensive study of the process of denaturation this question still remains unsettled. Moreover, its actuality has greatly increased lately with the accumulation of contradictory data.     

Heat of denaturation of lysozyme

The enthalpy of denaturation of lysozyme was determined by measuring the heat, capacity of an aqueous solution of this protein in the vicinity of the transition temperature, 46 °C at pH 1. Within experimental error the calorimetric, heat (56 ± 8 kcal/mole) was found to agree with the van't Hoff transition enthalpy (63 ± 6 kcal/mole) determined from optical rotation measurements as a function of temperature. This indicates that denaturation of this protein can be interpreted in terms of a two-state model. Successive measurements of the same sample showed, from several lines of evidence, that the transition was about 80% reversible for the particular environmental conditions and thermal history involved in the study.     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence