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Start Over Creator Kubala, M.

1. Fluorescence Competition Assay for the Assessment of ATP Binding to an Isolated Domain of Na+, K+-ATPase

Creator:
Kubala, M., Plášek, J., and Amler, E.
Type:
article, model:article, and TEXT
Subject:
Competition, Dissociation constant, Fluorescence, Enzyme-ligand interaction, and TNP-ATP
Language:
English
Description:
An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K+-ATPase. The estimated value of KD (6.2 ± 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public

2. Protein Modeling Combined with Spectroscopic Techniques: an Attractive Quick Alternative to Obtain Structural Information

Creator:
Kubala, M., Obšil, T., Obšilová, V., Lánský, Z., and Amler, E.
Type:
article, model:article, and TEXT
Subject:
Computer modeling, Molecular dynamics simulations, Fluorescence spectroscopy, Time-resolved fluorescence spectroscopy, Na+/K+-ATPase, 14-3-3 proteins, and Point mutations
Language:
English
Description:
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys 501, Gly502 and Arg544, also Glu446, Phe475 and Gln 482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth β-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.
Rights:
http://creativecommons.org/licenses/by-nc-sa/4.0/ and policy:public