Kinetic studies of cAMP-induced propagation of the allosteric signal in the cAMP receptor protein from Escherichia coli with the use of site-directed mutagenesis

Górecki, Andrzej; Kepys, Barbara; Bonarek, Piotr; Wasylewski, Zygmunt

doi:10.1016/j.ijbiomac.2008.12.015

Cited by 6 publications

(9 citation statements)

References 32 publications

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“…For example the single amino acid substitution in the hinge region between the domain of the bacterial transcription factor cAMP receptor protein substantially alters the reorientation of domains, rendering the apo‐protein alike holo form, or preventing its structural transformation into the holo form upon ligand binding, depending on the mutations introduced . Also, the kinetics of the signal transduction is substantially changed in these cases . Similarly, for the human transcription factor IIB, naturally occurring in two structural forms remaining in dynamic equilibrium, point mutations causing a shift of equilibrium towards a closed form incapable of binding molecular partner have been identified .…”

Section: Resultsmentioning

confidence: 99%

Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA – thermodynamic studies

Nieborak

Górecki

2016

FEBS Letters

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Edited by Alfonso ValenciaThis work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF.

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Section: Resultsmentioning

confidence: 99%

Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA – thermodynamic studies

Nieborak

Górecki

2016

FEBS Letters

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“…For example, a recently proposed mechanism for cyclic AMP receptor protein (CRP) and cAMP association suggests that two independent binding processes preceds a subsequent three step conformational changes. In this case, if more emphasis is given to the data content at initial stages, where binding process dominates, the effect of non-specific perturbations caused by conformational changes can be eliminated [38].…”

Section: Resultsmentioning

confidence: 99%

Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Krishnamoorthy

Mok

2010

PLoS ONE

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BackgroundNuclear Magnetic Resonance (NMR) spectroscopy offers a variety of experiments to study protein-ligand interactions at atomic resolution. Among these experiments, N Heteronuclear Single Quantum Correlation (HSQC) experiment is simple, less time consuming and highly informative in mapping the binding site of the ligand. The interpretation of N HSQC becomes ambiguous when the chemical shift perturbations are caused by non-specific interactions like allosteric changes and local structural rearrangement. Under such cases, detailed chemical exchange analysis based on chemical shift perturbation will assist in locating the binding site accurately.Methodology/Principal FindingsWe have automated the mapping of binding sites for fast chemical exchange systems using information obtained from N HSQC spectra of protein serially titrated with ligand of increasing concentrations. The automated program Auto-FACE (Auto-FAst Chemical Exchange analyzer) determines the parameters, e.g. rate of change of perturbation, binding equilibrium constant and magnitude of chemical shift perturbation to map the binding site residues. Interestingly, the rate of change of perturbation at lower ligand concentration is highly sensitive in differentiating the binding site residues from the non-binding site residues. To validate this program, the interaction between the protein and the ligand BH3I-1 was studied. Residues in the hydrophobic BH3 binding groove of were easily identified to be crucial for interaction with BH3I-1 from other residues that also exhibited perturbation. The geometrically averaged equilibrium constant () calculated for the residues present at the identified binding site is consistent with the values obtained by other techniques like isothermal calorimetry and fluorescence polarization assays (). Adjacent to the primary site, an additional binding site was identified which had an affinity of 3.8 times weaker than the former one. Further NMR based model fitting for individual residues suggest single site model for residues present at these binding sites and two site model for residues present between these sites. This implies that chemical shift perturbation can represent the local binding event much more accurately than the global binding event.Conclusion/SignificanceDetail NMR chemical shift perturbation analysis enabled binding site residues to be distinguished from non-binding site residues for accurate mapping of interaction site in complex fast exchange system between small molecule and protein. The methodology is automated and implemented in a program called “Auto-FACE”, which also allowed quantitative information of each interaction site and elucidation of binding mechanism.

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“…Selective labeling of a single cysteine residue or dual labeling within multicysteine proteins is a challenge, especially if the remaining cysteine residues are reactive and should remain in reduced form after labeling. In case of globular proteins, it may be slightly easier if some of the cysteine residues are located within the core of the protein structure, which renders them inaccessible for the dye . For surface cysteines, reactivity does not depend only on the steric effect, that is, on the accessibility of the dye, but also on electrostatic interactions between the reactive cysteinyl anion and the adjacent amino acids .…”

Section: Discussionmentioning

confidence: 99%

Site‐directed fluorescence labeling of intrinsically disordered region of human transcription factor YY1: The inhibitory effect of zinc ions

Górka

Górecki

Dziedzicka-Wasylewska

2017

Protein Science

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Site-specific labeling of proteins with fluorescent dyes allows the study of protein structure and function using a wide variety of fluorescent techniques. However, specific labeling is not trivial in the case of proteins containing multiple cysteine residues. An example of such a protein is transcription factor Yin Yang 1, which comprises eight cysteine residues in four C2H2 type zinc fingers in the C-terminal region. Kinetic measurements of the labeling process allowed us to develop preparative labeling of three cysteine residues differently introduced to the N-terminal, disordered fragment of the protein. The protocol developed in the present study allows to prepare the protein with high recovery yield and high selectivity of the labeling. This was confirmed using proteolytic digestion and spectroscopic approach. The labeling process was significantly affected by the presence of zinc ions and was dependent on the localization of the engineered cysteine residue. This is the first known example of the use of cysteine metal protection and labeling (CyMPL) technology for the labeling of protein regions with no stable secondary structures.

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Kinetic studies of cAMP-induced propagation of the allosteric signal in the cAMP receptor protein from Escherichia coli with the use of site-directed mutagenesis

Cited by 6 publications

References 32 publications

Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA – thermodynamic studies

Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA – thermodynamic studies

Auto-FACE: An NMR Based Binding Site Mapping Program for Fast Chemical Exchange Protein-Ligand Systems

Site‐directed fluorescence labeling of intrinsically disordered region of human transcription factor YY1: The inhibitory effect of zinc ions

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