Conformational Drift of Lactate Dehydrogenase

King, Lan; Weber, Gregorio

doi:10.1016/s0006-3495(86)83597-4

Cited by 12 publications

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“…The loss of free energy by the separated monomers is a slow process in comparison with the fast association-dissociation cycle (AD cycle). As a result the macroscopic equilibrium is reached very slowly (Xu & Weber, 1981; King & Weber, 1986c) and there may be difficulty in deciding whether one is measuring the final equilibrium value.…”

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Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure

Weber¹

1986

Biochemistry

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The native conformation of oligomers may be expected to undergo reversible changes when they separate upon dissociation of the original aggregate. When these changes are slow in comparison with the time of an association-dissociation (AD) cycle, they give rise to characteristic effects in the dependence of the dissociation: upon dilution, at constant pressure, and upon the applied pressure, at constant concentration. The phenomenological description of these effects is examined by comparing two possible models: The first model assumes a continuous loss in free energy of association with the extent of dissociation; the second supposes the existence of two or more distinct aggregates differing in subunit affinity and present in proportions that vary with the extent of dissociation. The latter model fits better the experimental data available, with regard to both the concentration and the pressure dependence of the association, and gives a particularly simple explanation of the hysteresis phenomena observed in several oligomeric proteins after application of pressure. The validity of the principle of detailed balance, often assumed in dealing with complex equilibria, is discussed in detail as it does not appear possible to reconcile it with some of the experimental observations or with the proposed model.

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Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure

Weber¹

1986

Biochemistry

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“…We have no expertise in the protein that is the focus of the work of Rajagopalan et al, but we note that G. Weber and collaborators characterized several proteins that dissociate when diluted over concentration ranges significantly smaller than the values predicted by simple equilibrium considerations. ,− Weber explained these observations in terms of what he called a “conformational drift”, where the increase in dissociation constant with dilution arises from a slow change in conformation of the monomers when they become separated from each other. We stress that we are not in a position to elaborate on whether this concept describes Rajagopalan’s data, or even if it does, whether it has any implications or relevance in terms of the biochemical function of the protein.…”

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confidence: 95%

Protein Oligomerization Equilibria and Kinetics Investigated by Fluorescence Correlation Spectroscopy: A Mathematical Treatment

Kanno

Levitus

2014

J. Phys. Chem. B

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Fluorescence correlation spectroscopy (FCS) is a technique that is increasingly being used to investigate protein oligomerization equilibria and dynamics. Each individual FCS decay is characterized by its amplitude and a characteristic diffusion time, both of which are sensitive to the degree of dissociation of the protein. Here, we provide a mathematical treatment that relates these observables with the parameters of interest: the equilibrium constants of the different protein dissociation steps and their corresponding dissociation and association kinetic rate constants. We focused on the two most common types of protein homooligomers (dimers and tetramers) and on the experimental variables relevant for the design of the experiment (protein concentration, fractional concentration of labeled protein). The analysis of the theoretical expectations for proteins with different dissociation constants is a key aspect of experiment design and data analysis and cannot be performed without a physically accurate treatment of the system. In particular, we show that the analysis of FCS data using some commonly used empirical models may result in a serious misinterpretation of the experimental results.

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“…The final model system studied here is L-lactate dehydrogenase (LDH, Sus scrofa), a homotetrameric protein that also dissociates under cold high-pressure conditions. 21 At ambient pressure (4ºC), LDH elutes as a broadened peak preceded by a small shoulder of aggregates or higher oligomers (Figure 6A). The estimated molecular weight is constant across the peak and closely matches the known molecular weight of the LDH homotetramer (149.61 kDa) 22 At 100 MPa (4ºC), an additional peak appears in the elution profile (Figure 6B) and the estimated molecular weight falls appreciably, though not fully to the expected dimer value (74.8 kDa).…”

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“…Enolase is a member of the former category since it shows rapid, reversible dissociation with little evidence of change in conformation 18 . L-lactate dehydrogenase, on the other hand, is a member of the latter category since it shows well-documented hysteresis with pressure that has been interpreted as conformational shift preventing rapid reassociation upon depressurization 21 . The differences in these two examples are very clear in HP-SEC-SAXS: upon pressurization, enolase becomes a mixture of dimer and monomer, but it remains a single peak in the chromatogram.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Inline SAXS-coupled chromatography under extreme hydrostatic pressure

Miller

Cummings

Huang

et al. 2022

Preprint

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As continuing discoveries highlight the surprising abundance and resilience of deep ocean and subsurface microbial life, the effects of extreme hydrostatic pressure on biological structure and function have attracted renewed interest. Biological small angle X-ray scattering (BioSAXS) is a widely used method of obtaining structural information from biomolecules in solution under a wide range of solution conditions. Due to its ability to reduce radiation damage, remove aggregates, and separate monodisperse components from complex mixtures, size-exclusion chromatography coupled SAXS (SEC-SAXS) is now the dominant form of BioSAXS at many synchrotron beamlines. While BioSAXS can currently be performed with some difficulty under pressure with non-flowing samples, it has not been clear how, or even if, continuously flowing SEC-SAXS, with its fragile media-packed columns, might work in an extreme high-pressure environment. Here we show, for the first time, that reproducible chromatographic separations coupled directly to high-pressure BioSAXS can be achieved at pressures up to at least 100 MPa and that pressure-induced changes in folding and oligomeric state and other properties can be observed. The apparatus described here functions at a range of temperatures (0° C - 50° C), expanding opportunities for understanding biomolecular rules of life in deep ocean and subsurface environments.

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Conformational Drift of Lactate Dehydrogenase

Cited by 12 publications

References 2 publications

Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure

Phenomenological description of the association of protein subunits subjected to conformational drift. Effects of dilution and of hydrostatic pressure

Protein Oligomerization Equilibria and Kinetics Investigated by Fluorescence Correlation Spectroscopy: A Mathematical Treatment

Inline SAXS-coupled chromatography under extreme hydrostatic pressure

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