Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Pagano, Livia; Toto, Angelo; Malagrinò, Francesca; Visconti, Lorenzo; Jemth, Per; Gianni, Stefano

doi:10.3390/ijms22020828

Cited by 23 publications

(27 citation statements)

References 58 publications

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“…As shown in Table , the double mutant cycle analysis − of double mutations demonstrates nonadditive properties upon substrate binding. The effect of G121V and T113V mutation overwhelms that of G121S and M42F.…”

Section: Results and Discussionmentioning

confidence: 99%

“…The weaker the NADPH binding, the smaller the 440 nm fluorescence increment observed in the titration experiments. As shown in Scheme and Table , the double mutant cycle analysis − of double mutations demonstrates nonadditive properties upon substrate binding. In general, the first point mutation affects strongly the NADPH binding compared to the second one (Δ G 1 > Δ G ′ 1 and Δ G 2 > Δ G ′ 2 ). …”

Section: Results and Discussionmentioning

confidence: 99%

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Effects of Distal Mutations on Ligand-Binding Affinity in E. coli Dihydrofolate Reductase

et al. 2021

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Mutations far from the center of chemical activity in dihydrofolate reductase (DHFR) can affect several steps in the catalytic cycle. Mutations at highly conserved positions and the distal distance of the catalytic center were designed, including single-point and double-point mutations. Upon ligand binding, the fluorescence of the intrinsic optical probe, tryptophan, decreases due to either fluorescence quenching or energy transfer. We demonstrated an optical approach in measuring the equilibrium dissociation constant for enzyme−cofactor, enzyme−substrate, and enzyme−product complexes in wildtype ecDHFR and each mutant. We propose that the effects of these distal mutations on ligand-binding affinity stem from the spatial steric hindrance, the disturbance on the hydrogen network, or the modification of the protein flexibility. The modified N-terminus tag in DHFR acts as a cap on the entrance of the substrate-binding cavity, squeezes the adenosine binding subdomain, and influences the binding of NADPH in some mutants. If the mutation positions are away from the N-terminus tag and the adenosine binding subdomain, the additive effects due to the Nterminus tag were not observed. In the double-mutant-cycle analysis, double mutations show nonadditive properties upon either cofactor or substrate binding. Also, in general, the first point mutation strongly affects the ligand binding compared to the second one.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Effects of Distal Mutations on Ligand-Binding Affinity in E. coli Dihydrofolate Reductase

et al. 2021

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“…The so-called ‘double mutant cycles’ methodology is a powerful approach to measuring, directly, the extent of energetic communication between interacting side-chains. The approach is founded on the synergic employment of site-directed mutagenesis and quantitative measurement of the biophysical properties of a protein system [ 59 , 60 , 61 , 62 ]. A double-mutant cycle postulates that mutations insisting on two non-interacting residues would return an additive effect.…”

Section: Interaction Between Protein Domains—supertertiary Structurementioning

confidence: 99%

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

et al. 2022

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The vast majority of our current knowledge about the biochemical and biophysical properties of proteins derives from in vitro studies conducted on isolated globular domains. However, a very large fraction of the proteins expressed in the eukaryotic cell are structurally more complex. In particular, the discovery that up to 40% of the eukaryotic proteins are intrinsically disordered, or possess intrinsically disordered regions, and are highly dynamic entities lacking a well-defined three-dimensional structure, revolutionized the structure–function paradigm and our understanding of proteins. Moreover, proteins are mostly characterized by the presence of multiple domains, influencing each other by intramolecular interactions. Furthermore, proteins exert their function in a crowded intracellular milieu, transiently interacting with a myriad of other macromolecules. In this review we summarize the literature tackling these themes from both the theoretical and experimental perspectives, highlighting the effects on protein folding and function that are played by (i) flanking disordered tails; (ii) contiguous protein domains; (iii) interactions with the cellular environment, defined as quinary structures. We show that, in many cases, both the folding and function of protein domains is remarkably perturbed by the presence of these interactions, pinpointing the importance to increase the level of complexity of the experimental work and to extend the efforts to characterize protein domains in more complex contexts.

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“…In addition to interactions with primary structure, secondary and tertiary structural features can be derived from measurements of double or higher order mutations by examining epistasis using double-mutant cycles (Horovitz, 1996;Pagano et al, 2021). In general, epistasis between two residues indicates structural collaboration, e.g., interaction (Horovitz, 1996;Pagano et al, 2021). For high epistasis, the effect of a double mutation is different from the summed effect of individual mutations.…”

Section: Reviewmentioning

confidence: 99%

Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics

2022

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Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Cited by 23 publications

References 58 publications

Effects of Distal Mutations on Ligand-Binding Affinity in E. coli Dihydrofolate Reductase

Effects of Distal Mutations on Ligand-Binding Affinity in E. coli Dihydrofolate Reductase

On the Effects of Disordered Tails, Supertertiary Structure and Quinary Interactions on the Folding and Function of Protein Domains

Exploring molecular biology in sequence space: The road to next-generation single-molecule biophysics

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