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1. New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin

   https://doi.org/10.1242/jeb.242615  

   Bautista, Naim M.; Malte, Hans; Natarajan, Chandrasekhar; Wang, Tobias; Storz, Jay F.; Fago, Angela (August 2021, Journal of Experimental Biology)

   ABSTRACT Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in red blood cells upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here, we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on purified Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease O2 saturation of Hb. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas diffusion chamber, we observed similar effects in Hbs of a phylogenetically diverse set of other caiman, alligator and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80–100 million years ago in the common ancestor of crocodilians. Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where the build-up of erytrocytic CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate. 

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2. Effect of NH2-terminal acetylation on the oxygenation properties of vertebrate haemoglobin

   https://doi.org/10.1042/BCJ20200623  

   Natarajan, Chandrasekhar; Signore, Anthony V.; Kumar, Vikas; Weber, Roy E.; Fago, Angela; Storz, Jay F. (October 2020, Biochemical Journal)

   null (Ed.)

   In vertebrate haemoglobin (Hb), the NH2-terminal residues of the α- and β-chain subunits are thought to play an important role in the allosteric binding of protons (Bohr effect), CO2 (as carbamino derivatives), chloride ions, and organic phosphates. Accordingly, acetylation of the α- and/or β-chain NH2-termini may have significant effects on the oxygenation properties of Hb. Here we investigate the effect of NH2-terminal acetylation by using a newly developed expression plasmid system that enables us to compare recombinantly expressed Hbs that are structurally identical except for the presence or absence of NH2-terminal acetyl groups. Experiments with native and recombinant Hbs of representative vertebrates reveal that NH2-terminal acetylation does not impair the Bohr effect, nor does it significantly diminish responsiveness to allosteric cofactors, such as chloride ions or organic phosphates. These results suggest that observed variation in the oxygenation properties of vertebrate Hbs is principally explained by amino acid divergence in the constituent globin chains rather than post-translational modifications of the globin chain NH2-termini. 

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3. When are two hydrogen bonds better than one? Accurate first-principles models explain the balance of hydrogen bond donors and acceptors found in proteins

   https://doi.org/10.1039/D0SC05084A  

   Vennelakanti, Vyshnavi; Qi, Helena W.; Mehmood, Rimsha; Kulik, Heather J. (January 2021, Chemical Science)

   null (Ed.)

   Hydrogen bonds (HBs) play an essential role in the structure and catalytic action of enzymes, but a complete understanding of HBs in proteins challenges the resolution of modern structural ( i.e. , X-ray diffraction) techniques and mandates computationally demanding electronic structure methods from correlated wavefunction theory for predictive accuracy. Numerous amino acid sidechains contain functional groups ( e.g. , hydroxyls in Ser/Thr or Tyr and amides in Asn/Gln) that can act as either HB acceptors or donors (HBA/HBD) and even form simultaneous, ambifunctional HB interactions. To understand the relative energetic benefit of each interaction, we characterize the potential energy surfaces of representative model systems with accurate coupled cluster theory calculations. To reveal the relationship of these energetics to the balance of these interactions in proteins, we curate a set of 4000 HBs, of which >500 are ambifunctional HBs, in high-resolution protein structures. We show that our model systems accurately predict the favored HB structural properties. Differences are apparent in HBA/HBD preference for aromatic Tyr versus aliphatic Ser/Thr hydroxyls because Tyr forms significantly stronger O–H⋯O HBs than N–H⋯O HBs in contrast to comparable strengths of the two for Ser/Thr. Despite this residue-specific distinction, all models of residue pairs indicate an energetic benefit for simultaneous HBA and HBD interactions in an ambifunctional HB. Although the stabilization is less than the additive maximum due both to geometric constraints and many-body electronic effects, a wide range of ambifunctional HB geometries are more favorable than any single HB interaction. 

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4. Quantum Mechanical Assessment of Protein–Ligand Hydrogen Bond Strength Patterns: Insights from Semiempirical Tight-Binding and Local Vibrational Mode Theory

   https://doi.org/10.3390/ijms24076311  

   Madushanka, Ayesh; Moura, Renaldo T.; Verma, Niraj; Kraka, Elfi (April 2023, International Journal of Molecular Sciences)

   Hydrogen bonds (HB)s are the most abundant motifs in biological systems. They play a key role in determining protein–ligand binding affinity and selectivity. We designed two pharmaceutically beneficial HB databases, database A including ca. 12,000 protein–ligand complexes with ca. 22,000 HBs and their geometries, and database B including ca. 400 protein–ligand complexes with ca. 2200 HBs, their geometries, and bond strengths determined via our local vibrational mode analysis. We identified seven major HB patterns, which can be utilized as a de novo QSAR model to predict the binding affinity for a specific protein–ligand complex. Glycine was reported as the most abundant amino acid residue in both donor and acceptor profiles, and N–H⋯O was the most frequent HB type found in database A. HBs were preferred to be in the linear range, and linear HBs were identified as the strongest. HBs with HB angles in the range of 100–110°, typically forming intramolecular five-membered ring structures, showed good hydrophobic properties and membrane permeability. Utilizing database B, we found a generalized Badger’s relationship for more than 2200 protein–ligand HBs. In addition, the strength and occurrence maps between each amino acid residue and ligand functional groups open an attractive possibility for a novel drug-design approach and for determining drug selectivity and affinity, and they can also serve as an important tool for the hit-to-lead process. 

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5. Parameter sensitivity analysis for CO-mediated sickle cell de-polymerization

   https://doi.org/10.55630/j.biomath.2023.12.036  

   Liu, Liping; Basti, Mufeed; Messan, Yao; Tang, Guoqing; Luke, Nicholas (March 2024, BIOMATH)

   This study investigates the impact of melting/binding rates (referred to hereafter as the parameters) over the polymers and monomers on the dynamics of carbon-monoxide-mediated sickle cell hemoglobin (HbS) de-polymerization. Two approaches, namely the traditional sensitivity analysis (TSA) and the multi-parameter sensitivity analysis (MPSA), have been developed and applied to the mathematical model system to quantify the sensitivities of polymers and monomers to the parameters. The Runge-Kutta method and the Monte-Carlo simulation are employed for the implementation of the sensitivity analyses. The TSA utilizes the traditional sensitivity functions (TSFs). The MPSA enumerates the overall effect of the model input parameters on the output by perturbing the model input parameters simultaneously within large ranges. All four concentrations (namely, de-oxy HbS monomers, CO-bound HbS monomers, de-oxy Hbs polymer and CO-bound HbS polymer) as model outputs, and all four binding/melting rates (namely, the CO binding and melting rates for polymers and monomers) as input parameters are considered in this study. The sensitivity results suggest that TSA and MPSA are essentially consistent. 

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