Isothermal denaturation fluorimetry vs differential scanning fluorimetry as tools for screening of stabilizers for protein freeze-drying: Human phenylalanine hydroxylase as the case study

Leandro, Paula; Lino, Paulo Roque; Lopes, Raquel; Leandro, João; Amaro, Mariana; Sousa, Paulo Gleisson Rodrigues de; Vicente, João B.; Almeida, António

doi:10.1016/j.ejpb.2023.03.012

Cited by 1 publication

(2 citation statements)

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“…The decrease in monomeric and dimeric states upon heating suggests that the structural fragments are involved in the formation of large insoluble aggregates. The hydrodynamic diameter, as determined by DLS, displayed values of 11.44 ± 0.24 nm for the native protein and 14.40 ± 2.37 nm for ALDH Tt -508, which is in accordance with the diameter reported for other globular proteins of similar molecular weight and with the predicted molecular weight using the Zetasizer Nano-ZSP instrument (264 and 212 kDa, respectively) . The values of experimentally derived D H reported are a weighted average obtained from the Zetasizer Nano-ZSP instrument, which creates a bias toward larger (aggregate) populations .…”

Section: Resultssupporting

confidence: 82%

“…The hydrodynamic diameter, as determined by DLS, displayed values of 11.44 ± 0.24 nm for the native protein and 14.40 ± 2.37 nm for ALDH Tt -508, which is in accordance with the diameter reported for other globular proteins of similar molecular weight and with the predicted molecular weight using the Zetasizer Nano-ZSP instrument (264 and 212 kDa, respectively). 23 The values of experimentally derived D H reported are a weighted average obtained from the Zetasizer Nano-ZSP instrument, which creates a bias toward larger (aggregate) populations. 24 The higher hydrodynamic diameter reported for ALDH Tt -508 is therefore likely to be caused by the higher presence of aggregates in the sample (5.9% as measured by SE-HPLC) which were not filtered ( D H < 0.22 μm).…”

Section: Resultsmentioning

confidence: 99%

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Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27

Brytan,

Shortall,

Duarte

et al. 2024

Biochemistry

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Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein's pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a Cterminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.

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Section: Resultssupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%