Asynchronous ligand binding and proton release in a root effect hemoglobin.

Ligand-binding equilibria, kinetics and 13C n.m.r. spectra of bound 13CO, of the haemoglobins from two fishes that are very distant on the evolutionary scale, i.e. the fourth haemoglobin component from Salmo irideus and the single component from Osteoglossum bicirrhosum, were studied. The C-terminal sequence was also determined for the haemoglobin from Osteoglossum. The results show that (i) the C-terminal residues of both chains are not directly responsible for the characteristic heterotropic effect known as Root effect, since for both fish haemoglobins these residues are identical with those of human haemoglobins. (ii) In all haemoglobins characterized by the Root effect a dependence of the 13CO n.m.r. resonances on pH is observed. However, the extent of the shift(s) depends on the particular protein, and is probably the result of a combination of both tertiary and quaternary conformational changes. (iii) Both haemoglobins from trout and Osteoglossum manifest a functional heterogeneity between the two types of chains in the tetramer, which increases with proton activity. For CO, the effect is very small for trout haemoglobin IV, and very marked for Osteoglossum haemoglobin; for O2 strongly heterogeneous binding curves were obtained at approx. pH6.2 with both haemoglobins. (iv) Estimations of the relative values of the affinity constants for the α and β chains in the tetramer were obtained for both haemoglobins from 13CO n.m.r. spectra at low fractional saturation. On the basis of these findings the molecular mechanism underlying the Root effect is discussed.

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The pH dependence of the affinity, kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

Comparative Biochemistry and Physiology Part B Comparative Biochemistry ◽

10.1016/0305-0491(90)90127-f ◽

1990 ◽

Vol 97 (4) ◽

pp. 815-820

Author(s):

T. Brittain ◽

R.M.G. Wells

Keyword(s):

Ligand Binding ◽

Carassius Auratus ◽

Ph Dependence ◽

Root Effect ◽

Goldfish Carassius Auratus

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A kinetic and equilibrium study of ligand binding to a Root-effect haemoglobin

Biochemical Journal ◽

10.1042/bj2280409 ◽

1985 ◽

Vol 228 (2) ◽

pp. 409-414 ◽

Cited By ~ 13

Author(s):

T Brittain

Keyword(s):

Ligand Binding ◽

Flash Photolysis ◽

State Model ◽

Hill Coefficient ◽

Affinity Constant ◽

Root Effect ◽

Equilibrium Study ◽

Titration Data ◽

Time Courses ◽

Two State Model

The blood of the striped marlin (Tetrapturus audax) contains one major Root-effect haemoglobin. Titrations of this haemoglobin with CO show that at high pH the molecule is highly co-operative (Hill coefficient 2.8) whereas at low pH the titration data can best be described as the sum of contributions from non-co-operating subunits of different affinity. In terms of the two-state model the R-state affinity constant is much more sensitive to pH than is that of the T state. Flash-photolysis studies were used to characterize the kinetics of ligand binding to this haemoglobin. Both T and R states show kinetic heterogeneity in their recombination time courses, associated with the alpha- and beta-chains of the molecule. The rate constants for ligand binding to each chain, in each quaternary state, were determined, and in conjunction with the allosteric equilibrium parameters determined at pH8.0 were used in the two-state analysis of reaction curves, over a range of ligand concentration. The two-state model, extended to take account of chain difference, adequately fits the homotrophic effects observed for this haemoglobin. The two-state model is, however, inadequate in its description of the heterotropic effects produced by protons.

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Biophysical Approaches Determining Ligand Binding to Biomolecular Targets

10.1039/9781849732666 ◽

2011 ◽

Cited By ~ 6

Keyword(s):

Ligand Binding

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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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