| 1. |
- Bosco, Daryl A, et al.
(författare)
-
Dissecting the microscopic steps of the cyclophilin a enzymatic cycle on the biological substrate HIV-capsid by NMR
- 2010
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 403:5, s. 723-38
-
Tidskriftsartikel (refereegranskat)abstract
- Peptidyl-prolyl isomerases (PPIases) are emerging as key regulators of many diverse biological processes. Elucidating the role of PPIase activity in vivo has been challenging because mutagenesis of active site residues not only reduces the catalytic activity of these enzymes, but also dramatically affects substrate binding. Employing the cyclophilin A (CypA) PPIase together with its biologically relevant and natively folded substrate, the N-terminal domain of the HIV-1 capsid (CA(N)) protein, we demonstrate here how to dissect residue specific contributions to PPIase catalysis versus substrate binding utilizing NMR spectroscopy. Surprisingly, a number of CypA active-site mutants previously assumed to be strongly diminished in activity toward biological substrates based on a peptide assay only, catalyze the HIV capsid with wild-type activity, but with a change in the rate-limiting step of the enzymatic cycle. The results illustrate that a quantitative analysis of catalysis using the biological substrates is critical when interpreting the effects of PPIase mutations in biological assays.
|
|
| 2. |
- Eisenmesser, Elan Zohar, et al.
(författare)
-
Enzyme dynamics during catalysis
- 2002
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 295:5559, s. 1520-1523
-
Tidskriftsartikel (refereegranskat)abstract
- Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory.
|
|
| 3. |
- Eisenmesser, Elan Z, et al.
(författare)
-
Intrinsic dynamics of an enzyme underlies catalysis
- 2005
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 438, s. 117-21
-
Tidskriftsartikel (refereegranskat)abstract
- A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis1, 2, 3, 4, 5. The concept of conformational substates has been described6; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis–trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously3, 7, 8, 9, 10, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.
|
|
| 4. |
- Gardino, Alexandra K, et al.
(författare)
-
Transient Non-native Hydrogen Bonds Promote Activation of a Signaling Protein
- 2009
-
Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 139:6, s. 1109-18
-
Tidskriftsartikel (refereegranskat)abstract
- SummaryPhosphorylation is a common mechanism for activating proteins within signaling pathways. Yet, the molecular transitions between the inactive and active conformational states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of a signaling protein, nitrogen regulatory protein C (NtrC), by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding and show that only a rarely populated, pre-existing active conformation is energetically stabilized by phosphorylation. Using nuclear magnetic resonance (NMR) dynamics, we test an atomic scale pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009). The data show that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results unravel atomistic details of native-state protein energy landscapes by expanding the knowledge about ground states to transition landscapes.
|
|
| 5. |
- Henzler-Wildman, Katherine A, et al.
(författare)
-
Intrinsic motions along an enzymatic reaction trajectory.
- 2007
-
Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 450, s. 838-44
-
Tidskriftsartikel (refereegranskat)abstract
- The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.
|
|
| 6. |
- Wolf-Watz, Magnus, et al.
(författare)
-
Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair
- 2004
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 11, s. 945-9
-
Tidskriftsartikel (refereegranskat)abstract
- A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase during catalysis. A dynamic process, the opening of the nucleotide-binding lids, was found to be rate-limiting for both enzymes as measured by NMR relaxation. Moreover, we found that the reduced catalytic activity of the hyperthermophilic enzyme at ambient temperatures is caused solely by a slower lid-opening rate. This comparative and quantitative study of activity, structure and dynamics revealed a close link between protein dynamics and catalytic turnover.
|
|
| 7. |
- Young, Iris D., et al.
(författare)
-
Structure of photosystem II and substrate binding at room temperature
- 2016
-
Ingår i: Nature. - : Macmillan Publishers Ltd.. - 0028-0836 .- 1476-4687. ; 540:7633, s. 453-457
-
Tidskriftsartikel (refereegranskat)abstract
- Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, in which S1 is the dark-stable state and S3 is the last semi-stable state before O–O bond formation and O2 evolution2,3. A detailed understanding of the O–O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site4–6. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL7 provided a damage-free view of the S1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10–13. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O–O bond formation mechanisms.
|
|
