Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase

Luk, Louis Y. P.; Ruiz‐Pernía, J. Javier; Adesina, Aduragbemi S.; Loveridge, E. Joel; Tuñón, Iñaki; Moliner, Vicent; Allemann, Rudolf Konrad

doi:10.1002/anie.201503968

Cited by 38 publications

(74 citation statements)

References 45 publications

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“…33 However, these studies show different effects of isotopic substitution depending on the enzymatic system. Beyond assessing the importance of protein dynamics in enzyme catalysis 34-36 heavy enzyme studies have also been used in the development of inhibitors for pharmaceutical applications as well. 37,38 Nevertheless, further investigation is vital to obtain a comprehensive understanding of the correlation between altered fast protein dynamics and the catalyzed reaction along with other effects of isotopic substitution on enzyme catalysis.…”

Section: Introductionmentioning

confidence: 99%

Protein Mass Effects on Formate Dehydrogenase

et al. 2017

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Isotopically labeled enzymes (denoted as “heavy” or “Born Oppenheimer” enzymes) have been used to test the role of protein dynamics in catalysis. The original idea was that the protein's higher mass would reduce the frequency of its normal-modes without altering its electrostatics. Heavy enzymes have been used to test if the vibrations in the native enzyme are coupled to the chemistry it catalyzes, and different studies have resulted in ambiguous findings. Here the temperature-dependence of intrinsic kinetic isotope effects of the enzyme formate dehydrogenase is used to examine the distribution of H-donor to H-acceptor distance as a function of the protein's mass. The protein dynamics are altered in the heavy enzyme to diminish motions that determine the transition state sampling in the native enzyme, in accordance with a Born Oppenheimer-like effect on bond activation. Findings of this work suggest components related to fast frequencies that can be explained by Born-Oppenheimer enzyme hypothesis (vibrational) and also slower timescale events that are non-Born-Oppenheimer in nature (electrostatic), based on evaluations of protein mass dependence of donor-acceptor-distance and forward commitment to catalysis along with steady state and single turnover measurements. Together, the findings suggest that the mass modulation affected both local, fast, protein vibrations associated with the catalyzed chemistry, and the protein's macromolecular electrostatics at slower timescales, i.e., both Born Oppenheimer and non-Born Oppenheimer effects are observed. Comparison to previous studies leads to the conclusion that isotopic labeling of the protein may have different effects on different systems, however making heavy enzyme studies a very exciting technique for exploring the dynamics link to catalysis in proteins.

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Section: Introductionmentioning

confidence: 99%

Protein Mass Effects on Formate Dehydrogenase

et al. 2017

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“…Some recent examples include ubiquitin 36 , alpha-synuclein 37 , histones 38 , and membrane proteins 39 . Additional examples include fundamental ubiquitin biology 40 , proteins with selective isotopic labeling 41 , site-specific installation of fluors (e.g., FRET pairs) 42 , and interesting scaffold approaches 43 .…”

Section: Introductionmentioning

confidence: 99%

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Jacobsen

Erickson

Kay

2017

Bioorganic & Medicinal Chemistry

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The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

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“…These apparently contradictory results demonstrate the delicate nature of motional coupling to catalysis. We have refined this global picture of motional coupling by isotopically labeling specific segments of an enzyme and therefore experimentally determining which parts of the enzyme show motional coupling [19]. Two isotopic hybrids of DHFR were prepared by chemical ligation techniques [20], one in which the mobile N-terminal segment contained heavy isotopes while the remainder of the protein was of natural isotopic abundance, and one in which only the C-terminal region was isotopically labeled.…”

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confidence: 99%

“…Two isotopic hybrids of DHFR were prepared by chemical ligation techniques [20], one in which the mobile N-terminal segment contained heavy isotopes while the remainder of the protein was of natural isotopic abundance, and one in which only the C-terminal region was isotopically labeled. These experiments revealed that the N-terminal region is only involved in beneficial millisecond conformational motions, while in the C-terminal region fast dynamic motions couple unfavorably to barrier crossing, thereby slowing the reaction [19]. The combination of chemical ligation and heavy enzyme approaches has shown for the first time that different parts of an enzyme affect different aspects of its function.…”

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confidence: 99%