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1. The role of Ca ²⁺ and protein scaffolding in the formation of nature’s water oxidizing complex

   https://doi.org/10.1073/pnas.2011315117  

   Avramov, Anton P.; Hwang, Hong J.; Burnap, Robert L. (November 2020, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Photosynthetic O 2 evolution is catalyzed by the Mn 4 CaO 5 cluster of the water oxidation complex of the photosystem II (PSII) complex. The photooxidative self-assembly of the Mn 4 CaO 5 cluster, termed photoactivation, utilizes the same highly oxidizing species that drive the water oxidation in order to drive the incorporation of Mn 2+ into the high-valence Mn 4 CaO 5 cluster. This multistep process proceeds with low quantum efficiency, involves a molecular rearrangement between light-activated steps, and is prone to photoinactivation and misassembly. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn 2+ and Ca 2+ ions in genetically engineered membranes of the cyanobacterium Synechocystis sp. PCC6803 to elucidate the action of Ca 2+ and peripheral proteins. We show that the protein scaffolding organizing this process is allosterically modulated by the assembly protein Psb27, which together with Ca 2+ stabilizes the intermediates of photoactivation, a feature especially evident at long intervals between photoactivating flashes. The results indicate three critical metal-binding sites: two Mn and one Ca, with occupation of the Ca site by Ca 2+ critical for the suppression of photoinactivation. The long-observed competition between Mn 2+ and Ca 2+ occurs at the second Mn site, and its occupation by competing Ca 2+ slows the rearrangement. The relatively low overall quantum efficiency of photoactivation is explained by the requirement of correct occupancy of these metal-binding sites coupled to a slow restructuring of the protein ligation environment, which are jointly necessary for the photooxidative trapping of the first stable assembly intermediate. 

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2. The Mutant β ^E202K Sliding Clamp Protein Impairs DNA Polymerase III Replication Activity

   https://doi.org/10.1128/JB.00303-21  

   Homiski, Caleb; Scotland, Michelle K.; Babu, Vignesh M.; Chodavarapu, Sundari; Maul, Robert W.; Kaguni, Jon M.; Sutton, Mark D. (November 2021, Journal of Bacteriology)

   Silhavy, Thomas J. (Ed.)

   ABSTRACT Expression of the Escherichia coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro . Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β E202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, β E202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaN E202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β E202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than appreciated currently. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes Escherichia coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β E202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III, and IV. 

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3. Fast Magic‐Angle Spinning ¹⁹ F NMR Spectroscopy of HIV‐1 Capsid Protein Assemblies

   https://doi.org/10.1002/anie.201809060  

   Wang, Mingzhang; Lu, Manman; Fritz, Matthew P.; Quinn, Caitlin M.; Byeon, In‐Ja L.; Byeon, Chang‐Hyeock; Struppe, Jochem; Maas, Werner; Gronenborn, Angela M.; Polenova, Tatyana (October 2018, Angewandte Chemie International Edition)

   Abstract ¹⁹F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS)¹⁹F solid‐state NMR spectroscopy in assemblies of HIV‐1 capsid protein. Tryptophan residues with fluorine substitution at the 5‐position of the indole ring were used as the reporters. The¹⁹F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin‐diffusion and radio‐frequency‐driven‐recoupling experiments were performed at MAS frequencies of 35 kHz and 40–60 kHz, respectively. Fast MAS frequencies of 40–60 kHz are essential for consistently establishing¹⁹F–¹⁹F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS¹⁹F NMR spectroscopy for structural analysis in large biological assemblies. 

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4. Illuminating an invisible state of the HIV-1 capsid protein CTD dimer using ¹⁹ F NMR and weighted ensemble simulations

   https://doi.org/10.1073/pnas.2420371122  

   Yang, Darian T; Chong, Lillian T; Gronenborn, Angela M (February 2025, Proceedings of the National Academy of Sciences)

   The HIV-1 capsid protein (CA) assembles into a conical shell during viral maturation, encasing and protecting the viral RNA genome. The C-terminal domain (CTD) of the two-domain capsid protein dimerizes, and this dimer connects individual chains in the mature capsid lattice. Previous NMR studies have shown that different dimer arrangements can be formed by isolated capsid protein chains and in assembled capsid lattices; however, the dynamics and functional relevance of these alternate dimers are unknown. To explore the conformational landscape of the CA-CTD dimer, we carried out atomistic molecular dynamics simulations using the weighted ensemble path sampling strategy, generating an ensemble of conformations. Focusing on the two dimer forms previously observed via solution NMR, we refined the conformational ensemble to highlight two metastable states using a Markov state model. Experimentally, we measured the interconversion rates between the two alternate dimers using¹⁹F NMR, and these rates showed good agreement with the interconversion rates derived from the simulations. After identifying the key interactions that distinguish the dimer states, the alternate dimer was further experimentally verified through disulfide crosslinking. Our results demonstrate the advantages of pairing weighted ensemble path sampling with¹⁹F NMR to gain atomistic insights into the hidden dimer state of the HIV-1 capsid protein. 

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5. Directly Functionalized Cucurbit[7]uril as a Biosensor for the Selective Detection of Protein Interactions by ¹²⁹ Xe hyperCEST NMR

   https://doi.org/10.1002/chem.201900610  

   Truxal, Ashley E.; Cao, Liping; Isaacs, Lyle; Wemmer, David E.; Pines, Alexander (February 2019, Chemistry – A European Journal)