Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin.

Ahmad, Faizan; Bigelow, Charles C.

doi:10.1016/s0021-9258(18)33605-6

Cited by 179 publications

(90 citation statements)

References 16 publications

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“…D 2 O is known to have effects that are qualitatively opposite those of denaturants such as urea or guanidinium chloride, typically reducing protein solubility and increasing stability (59)(60)(61)(62)(63). To counter this effect, the samples used in this study included 1 M urea, a concentration that does not significantly destabilize the native conformations of BPTI or Mb (64)(65)(66) and causes only small changes in the SAXS profile for l N (51).…”

Section: Resultsmentioning

confidence: 99%

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Goldenberg

Argyle

2014

Biophysical Journal

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Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with (2)H, and the concentrations of D2O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.

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Section: Resultsmentioning

confidence: 99%

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Goldenberg

Argyle

2014

Biophysical Journal

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“…To counter this effect, urea was added to a concentration of 1 M in all of the samples used for the present study. Urea at this concentration does not significantly destabilize the native conformations of BPTI or Mb (38)(39)(40). SAXS measurements of Mb and BPTI solutions were carried out using a laboratory-scale instrument with a slitcollimated x-ray beam.…”

Section: Resultsmentioning

confidence: 99%

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Goldenberg

Argyle

2014

Biophysical Journal

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Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D2O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10(-4) cm(3)mol/g(2) for Mb in phosphate buffer, 1.6 ×10(-4) cm(3)mol/g(2) for BPTI in phosphate buffer and 9.2 ×10(-4) cm(3)mol/g(2) for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.

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“…Then, dimers resistance diminishes to zero for C D-H/Et in 6 M urea (Figure 3, right panel). Indeed, RNase A is known to have just started its unfolding in 6 M urea [47], and this could suggest that dimers dissociation is almost contemporary to enzyme unfolding.…”

Section: Discussionmentioning

confidence: 99%

RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure–Catalytic Properties and Antitumor Activity

Montioli

Campagnari

Fasoli

et al. 2021

Life

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Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth.

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Estimation of the free energy of stabilization of ribonuclease A, lysozyme, alpha-lactalbumin, and myoglobin.

Cited by 179 publications

References 16 publications

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure–Catalytic Properties and Antitumor Activity

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