Spectroscopic Methods for the Determination of Protein Interactions

Groemping, Yvonne; Hellmann, Nadja

doi:10.1002/0471140864.ps2008s39

Cited by 15 publications

(8 citation statements)

References 16 publications

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“…F A and F AS represent the fluorescence signal of free protein A (UBM2) and the complex AS (UBM2⅐Ub), respectively, C A is the total concentration of UBM2, C S is the total concentration of the substrate S (Ub), and p is the stoichiometry value (28).…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Conserved Ubiquitin-binding Motifs (UBMs) of the Translesion Polymerase iota in Complex with Ubiquitin

Burschowsky

Rudolf

Rabut

et al. 2011

Journal of Biological Chemistry

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Ubiquitin-binding domains (UBDs) provide specificity to the ubiquitin system, which is also involved in translesion synthesis (TLS) in eukaryotic cells. Upon DNA damage, the UBDs (UBM domains) of polymerase iota (Pol ) interact with ubiquitinated proliferating cell nuclear antigen to regulate the interchange between processive DNA polymerases and TLS. We report a biophysical analysis and solution structures of the two conserved UBM domains located in the C-terminal tail of murine Pol in complex with ubiquitin. The 35-amino acid core folds into a helix-turn-helix motif, which belongs to a novel domain fold. Similar to other UBDs, UBMs bind to ubiquitin on the hydrophobic surface delineated by Leu-8, Ile-44, and Val-70, however, slightly shifted toward the C terminus. In addition, UBMs also use electrostatic interactions to stabilize binding. NMR and fluorescence spectroscopy measurements revealed that UBMs bind monoubiquitin, and Lys-63-but not Lys-48-linked chains. Importantly, these biophysical data are supported by functional studies. Indeed, yeast cells expressing ubiquitin mutants specifically defective for UBM binding are viable but sensitive to DNA damaging conditions that require TLS for repair.Ubiquitin plays a major role in regulating diverse biological pathways by changing the function, localization, or turnover of target proteins. In most cases, ubiquitin gets attached to an ⑀-amine of a lysine side chain via an isopeptide bond (1) and serves as a molecular tag by providing an additional binding surface, which is recognized by a plethora of different ubiquitin-binding domains (UBDs) 6 from at least 16 different families (2, 3). Most UBDs use ␣-helical structures to bind a solvent-exposed hydrophobic patch on the ␤-sheet of ubiquitin, which includes Leu-8, Ile-44, and Val-70. The amino acids surrounding this hydrophobic patch are chemically diverse, thus allowing for different binding modes (2-4). Ubiquitin can be attached on target proteins either as single (monoubiquitination) or multiple single moieties (multiubiquitination), or as several ubiquitins covalently bound to each other in a chain-like fashion (polyubiquitination). These chains can be linked to the N terminus or any of the seven different lysines of ubiquitin; Lys-11 and Lys-48 are the most frequently used, whereas Lys-48 and Lys-63 are the best studied (5). How ubiquitin tags and chains are specifically recognized by the different UBDs remains an important but poorly understood question (2, 3).Linkage-dependent spatial conformations taken by different ubiquitin chains significantly contribute to the specificity of UBD-ubiquitin interactions. Some domains exclusively bind Lys-48-(6, 7) or Lys-63-linked ubiquitin chains (8). NMR spectroscopy revealed that individual ubiquitin moieties in Lys-63-linked chains resemble beads on a string, whereas ubiquitins in Lys-48-linked chains fold onto each other (9, 10). It remains unclear why some domains show a clear preference toward either form of ubiquitin chains. Moreover, the binding preference i...

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Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Conserved Ubiquitin-binding Motifs (UBMs) of the Translesion Polymerase iota in Complex with Ubiquitin

Burschowsky

Rudolf

Rabut

et al. 2011

Journal of Biological Chemistry

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“…HSA is a single chain protein that contains one tryptophan residue (Trp214). Since the emission of this aminoacid is very sensitive to changes in its microenvironment, its fluorescence can be used as a signal for monitoring interactions [14]. So, in binding assays the fluorescence intensities of HSA were determined by monitoring the changes in fluorescence intensities from Trp214.…”

Section: Determination Of Binding Parametersmentioning

confidence: 99%

Interaction between Human Serum Albumin and antidiabetic compounds and its influence on the O2(1Δ )-mediated degradation of the protein

Challier

Beassoni

Boetsch

et al. 2015

Journal of Photochemistry and Photobiology B: Biology

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The complexity depicted by disease scenarios as diabetes mellitus, constitutes a very interesting field of study when drugs and biologically relevant components may be affected by such environments. In this report, the interaction between the protein Human Serum Albumin (HSA) and two antidiabetics (Andb), Gliclazide (Gli) and Glipizide (Glip) was studied through fluorescence and docking assays, in order to characterize these systems. On the basis that HSA and Andb can be exposed in vivo at high Reactive Oxygen Species (ROS) concentrations in diabetic patients, the degradative process of the protein free and bound to Andb, in presence of the species singlet molecular oxygen (O2((1)Δg)), was evaluated. Fluorescence and docking assays indicated that Gli, as well as Glip bind to HSA on two sites, with binding constants values in the order of 10(4)-10(5)M(-1). Likewise, docking assays revealed that the location of Gli or Glip on the protein may be the HSA binding sites II and III. Thermodynamic parameters showed that the interaction between HSA and Glip is a favored, enthalpically-controlled process. Oxygen uptake experiments indicated that Glip is less photooxidizable than Gli through a O2((1)Δg)-mediated process. Besides, the protein-Andb binding produced a decrease in the overall rate constant for O2((1)Δg) quenching as compared to the value for the free protein. This fact could be interpreted in terms of a reduction in the availability of Tyrosine residues in the bonded protein, with a concomitant decrease in the physical quenching deactivation of the oxidative species.

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“…Various spectroscopic methods, also based on fluorescence emission, have been developed for studying protein-protein interactions (for details see Groemping and Hellmann [20]). Using fluorescence microscopy, Demeule et al [21] have shown how protein aggregates can be detected and char-acterised.…”

Section: Research Articlementioning

confidence: 99%

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Garidel

Hegyi

Bassarab

et al. 2008

Biotechnology Journal

100

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Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.

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Spectroscopic Methods for the Determination of Protein Interactions

Cited by 15 publications

References 16 publications

Structural Analysis of the Conserved Ubiquitin-binding Motifs (UBMs) of the Translesion Polymerase iota in Complex with Ubiquitin

Structural Analysis of the Conserved Ubiquitin-binding Motifs (UBMs) of the Translesion Polymerase iota in Complex with Ubiquitin

Interaction between Human Serum Albumin and antidiabetic compounds and its influence on the O2(1Δ )-mediated degradation of the protein

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

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