Protein Dynamics and the Evolution of Novel Protein Function

Zimmermann, Jörg; Thielges, Megan C.; Yu, Wayne; Romesberg, Floyd E.

doi:10.1002/9783527634026.ch6

Cited by 2 publications

(2 citation statements)

References 214 publications

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“…Regions distant from the active site are essential and play a role in efficient catalytic function through protein motions. ,,,,− Temperature-dependent HDX studies across diverse families of enzymes have found unique combinations of flexibility and rigidity that arise in spatially resolved ways and can be linked to active site chemistry. − From the results documented herein with the enzyme enolase, we propose that the primary loop closure within L1 is tightly coupled to the dynamics of several thermal networks that originate at the protein/solvent interface (Figure ). The closing of L1 fine-tunes the distribution of thermal activation that initiates catalysis via transient adjustments to the distance and electrostatic interactions between substrate and the active site base (region A in Figure ).…”

Section: Discussionmentioning

confidence: 88%

Identification of Thermal Conduits That Link the Protein–Water Interface to the Active Site Loop and Catalytic Base in Enolase

et al. 2021

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We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pK a of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen−deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein−solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.

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Section: Discussionmentioning

confidence: 88%

Identification of Thermal Conduits That Link the Protein–Water Interface to the Active Site Loop and Catalytic Base in Enolase

et al. 2021

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“…It is clear that the ideal vibrational reporter has not been developed to date. For example, while the C–D bond vibrational reporters 15, 20, 21 are the least likely to perturb the native protein structure, they have some of the shortest vibrational lifetimes. 22 On the other hand, metal carbonyls 5, 19, 23, 24 have the largest transition dipole strengths and intermediate values for vibrational lifetimes, but are also the bulkiest reporters sterically and therefore the most likely to cause perturbations of the native structure.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and evaluation of the sensitivity and vibrational lifetimes of thiocyanate and selenocyanate infrared reporters

et al. 2016

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Two novel 2′-deoxyadenosine (dA) analogues, Si2-dA-SCN and Si2-dA-SeCN, and two novel phenylalanine (Phe) analogues, Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN, have been synthesized and the thiocyanate (SCN) and selenocyanate (SeCN) functional groups evaluated as vibrational reporters. The syntheses of Si2-dA-SCN and Si2-dA-SeCN were accomplished in three steps in 16% and 32% overall yields, respectively, and the syntheses of Boc-Me-PheCH2SCN and Boc-Me-PheCH2SeCN were completed in four steps in 8.9% and 2.3% overall yields, respectively. The SCN and SeCN stretch vibrational modes were shown to be sensitive to the local environment by frequency shifts and full-width half-maximum (fwhm) changes in response to tetrahydrofuran (THF) and THF/water solvent mixtures. The vibrational lifetimes of the Si2-dA-SeCN (237±12 ps) and Boc-Me-PheCH2SeCN (295±31 ps) in THF solution were determined by ultrafast infrared pump-probe spectroscopy to be 1.5 to 3 times longer than those for Si2-dA-SCN (140±6 ps) and Boc-Me-PheCH2SCN (102±4 ps). The longer lifetimes for the SeCN analogues were attributed to the better insulating effects of the heavier selenium atom compared to the sulfur atom. The solvent sensitivity and longer vibrational lifetimes compared to other vibrational reporters suggest that SCN and SeCN vibrational reporters are well suited to studying several dynamic processes including protein and nucleic acid hydration and conformational changes, however stability issues may require post-synthetic modification methods to incorporate these reporters into biomacromolecules.

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Protein Dynamics and the Evolution of Novel Protein Function

Cited by 2 publications

References 214 publications

Identification of Thermal Conduits That Link the Protein–Water Interface to the Active Site Loop and Catalytic Base in Enolase

Identification of Thermal Conduits That Link the Protein–Water Interface to the Active Site Loop and Catalytic Base in Enolase

Synthesis and evaluation of the sensitivity and vibrational lifetimes of thiocyanate and selenocyanate infrared reporters

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