Proton NMR spectroscopy was applied to myoglobin in the ferric, water-liganded form (metMbH2O) and the apo form (apoMb) to probe the structure and stability of the latter. Proteins from sperm whale and horse skeletal muscles were studied to simplify the spectral assignment task. Nuclear Overhauser effects and the response of chemical shifts to variations of pH were used as indicators of residual native holoprotein structure in the apoprotein. The investigation was focused in the histidine side chains and their environment. In metMbH2O, the resonances of all imidazole rings not interacting with the heme were assigned by applying standard two-dimensional methods. These assignments were found to differ from those reported elsewhere [Carver, J. A., & Bradbury, J. H. (1984) Biochemistry 23, 4890-4905] except for His-12, -113, and -116. Only one histidine (His-36) has a pK(a) higher than 7, two (His-48 and His-113) have a pK(a) lower than 5.5, and two (His-24 and His-82) appear not to titrate between pH 5.5 and pH 10. In the apoproteins, the signals of His-113 and His-116, as well as those of His-24, -36, -48, and -119 previously assigned in the horse globin [Cocco, M. J.. & Lecomte, J. T. J. (1990) Biochemistry 29, 11067-11072], could be followed between pH 5 and pH 10. A comparison to the holoprotein data indicated that heme removal has limited effect on the pK(a) and the surroundings of these residues. Five additional histidines which occur in the two helices and connecting loops forming the heme binding site were identified in the horse apoprotein. Four of these were found to have pK(a) values lower than that expected of an exposed residue. The NOE and titration data were proposed to reflect the fact that several holoprotein structural elements, in particular outside the heme binding site, are maintained in the apoprotein. In the heme binding region of the apoprotein structure, the low pK(a)'s suggest local environments which are resistant to protonation.
Truncated hemoglobins (trHbs) are heme proteins found in bacteria, plants, and unicellular eukaryotes. They are distantly related to vertebrate hemoglobins and are typically shorter than these by 20-40 residues. The multiple amino acid deletions, insertions, and replacements result in distinctive alterations of the canonical globin fold and a wide range of chemical properties. An early phylogenetic analysis categorized trHbs into three groups, I (trHbN), II (trHbO), and III (trHbP). Here, we revisit this analysis with 111 trHbs. We find that trHbs are orthologous within each group and paralogous across the groups. Group I globins form the most disparate set and separate into two divergent subgroups. Group II is comparatively homogeneous, whereas Group III displays the highest level of overall conservation. In Group I and Group II globins, for which some ligand binding and structural data are available, an improved description of probable protein-ligand interactions is achieved. Other conservation trends are either confirmed (essential glycines in loops), refined (lining of ligand access tunnel), or newly identified (helix start signal). The Group III globins, so far uncharacterized, exhibit recognizable heme cavity residues while lacking some of the residues thought to be important to the trHb fold. An analysis of the phylogenetic trees of each group provides a plausible scenario for the emergence of trHbs, by which the Group II trHb gene was the original gene, and the Group I trHb and Group III trHb genes were obtained via duplication and transfer events.
The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.
In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.
Perturbation of pK a values can change the favored protonation states of the nucleobases at biological pH and thereby modulate the function of RNA and DNA molecules. In an effort to understand the driving forces for pK a shifting specific to nucleic acids, we developed a thermodynamic framework that relates proton binding to the nucleobases and the helix-coil transition. Key features that emerge from the treatment are a comprehensive description of all the actions of proton binding on RNA folding: acid and alkaline denaturation of the helix and pK a shifting in the folded state. Practical experimental approaches for measuring pK a s from thermal denaturation experiments are developed. Microscopic pk a values (where k a is the acid dissociation constant) for the unfolded state were determined directly by experiments on unstructured oligonucleotides, which led to a macroscopic pK a for the ensemble of unfolded states shifted toward neutrality. The formalism was then applied to pH-dependent UV melting data for model DNA oligonucleotides. Folded-state pk a values were in good agreement with the outcome of pH titrations, and the acid and alkaline denaturation regions were well described. The formalism developed here is similar to that of Draper and coworkers for Mg 2+ binding to RNA, except that the unfolded state is described explicitly owing to the presence of specific proton-binding sites on the bases. A principal conclusion is that it should be possible to attain large pK a shifts by designing RNA molecules that fold cooperatively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.