Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco

doi:10.1002/pro.379

Cited by 20 publications

(38 citation statements)

References 38 publications

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“…The possibility that they may act as dissociative inhibitors can be ruled out because fluorescence resonance energy transfer (FRET) experiments, performed as described in ref. 16, show that they do not promote dissociation of the hTS dimer into monomers (SI Appendix).…”

Section: Resultsmentioning

confidence: 99%

“…The protocol employed to conjugate the two probes to the dimeric protein, the structural aspects (the two probes bind at C43 and C43′) and the quantitative analysis of the steady-state fluorescence data are described in detail in ref. 16. Solutions of the diconjugated protein in phosphate buffer, pH 7.5, were checked at the UV-visible spectrophotometer (Varian Cary 100) to contain comparable concentrations (typically ca.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore the dimeric assembly is necessary for the catalytic activity. The hTS dimer-to-monomer dissociation constant (K d ) was recently reported to be 200 nM (16). The active and inactive forms differ in the conformation of the loop from residue 181 to residue 197, which contains the catalytic cysteine, residue 195.…”

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confidence: 99%

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Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Cardinale

Guaitoli

Tondi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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157

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The undersigned authors note the following: "We wish to bring to your attention an issue regarding our PNAS publication referenced above. Although we cite our earlier PNAS publication (see ref. Figs. 2 and 3 display the UWHBs for Hb β-subunit (pdb.1bz0, chain B) and human cellular prion protein (pdb.1qm0) (12)(13)(14). Within the natural interactive context of the Hb subunit, the UWHBs signal crucial binding regions (24): UWHBs (90, 94), (90, 95) are associated with the β-FG corner involved in the quaternary α1β2 interface; UWHB (5, 9) is adjacent to Glu-6 which in sickle cell anemia mutates to Val-6 and is located at the Val-6-(Phe-85, Leu-88) interface in the deoxyHbS fiber."The following text in the section titled 'Toward a Structural Diagnosis' on page 6449 of our text is similar to the text beginning in the last paragraph on page 2392 in ref. 23:The distribution of proteins according to their average extent of hydrogen bond wrapping and their spatial concentration of structural defects is shown in Fig. 5 (see also ref. 23). The sample of 2,811 PDB proteins is large enough to define a reliable abundance distribution with an inflection point at ρ = 6.20. The integration of the distribution over a ρ-interval gives the fraction of proteins whose ρ lies within that range. Of the 2,811 proteins examined, 2,572 have ρ > 6.20, and none of them is known to yield amyloid aggregation under physiological conditions entailing partial retention of structure. Strikingly, relatively few disease-related amyloidogenic proteins are known in the sparsely populated, underwrapped 3.5 < ρ < 6.20 range, with the cellular prion proteins located at the extreme of the spectrum (3.53 < ρ < 3.72)....The range of H-bond wrapping 3.5 < ρ < 4.6 of 20 sampled PDB membrane proteins has been included in Fig. 5 for comparison. As expected, such proteins do not have the stringent H-bond packing requirements of soluble proteins for their H bonds at the lipid interface. Thus, this comparison becomes suggestive in terms of elucidating the driving factor for aggregation in soluble proteins: Although the UWHB constitutes a structural defect in a soluble protein because of its vulnerability to water attack, it is not a structural defect in a membrane protein. The exposure of the polar amide and carbonyl of the unbound state to a nonpolar phase is thermodynamically unfavorable (22). The virtually identical ρ value for human prion and outer-membrane protein A (Fig. 5) is revealing in this regard.Furthermore, all known amyloidogenic proteins that occur naturally in complexed form have sufficient H-bond wrapping within their respective complexes (ρ value near 6.2). Their amyloidogenic propensity appears only under conditions in which the protein is dissociated from the complex (compare Fig. 5). This finding is corroborated by the following computation. If an intramolecular hydrogen bond is underwrapped within the isolated protein molecule but located at an interface upon complexation, then to determine its extent of wrapping within the complex, we take ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Cardinale

Guaitoli

Tondi

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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157

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“…Reference spectra were taken for solutions containing equal concentrations of DLPC liposomes and subtracted from the sample spectra. FRET efficiency was calculated using the following equation [31]…”

Section: Methodsmentioning

confidence: 99%

“…Assuming that the monomer-dimer equilibrium is not affected by the dye labeling of the protein, the following relationship is obtained [31]…”

Section: Methodsmentioning

confidence: 99%

EmrE dimerization depends on membrane environment

Dutta

Morrison

Henzler-Wildman

2014

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer-dimer equilibrium constant (KMD2D ) of 0.002–0.009 mol % for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the KMD2D is only 0.8–0.95 mol % (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes KMD2D is 0.0005–0.0008 mol % based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity.

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PDB_Amyloid: an extended live amyloid structure list from the PDB

2018

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The Protein Data Bank ( PDB ) contains more than 135 000 entries at present. From these, relatively few amyloid structures can be identified, since amyloids are insoluble in water. Therefore, most amyloid structures deposited in the PDB are in the form of solid state NMR data. Based on the geometric analysis of these deposited structures, we have prepared an automatically updated web server, which generates a list of the deposited amyloid structures, and also entries of globular proteins that have amyloid‐like substructures of given size and characteristics. We have found that by applying only appropriately selected geometric conditions, it is possible to identify deposited amyloid structures and a number of globular proteins with amyloid‐like substructures. We have analyzed these globular proteins and have found proof in the literature that many of them form amyloids more easily than many other globular proteins. Our results relate to the method of Stanković et al . [Stanković I et al . (2017) IPSI BgD Tran Int Res 13, 47–51], who applied a hybrid textual‐search and geometric approach for finding amyloids in the PDB . If one intends to identify a subset of the PDB for certain applications, the identification algorithm needs to be re‐run periodically, since in 2017 on average 30 new entries per day were deposited in the data bank. Our web server is updated regularly and automatically, and the identified amyloid and partial amyloid structures can be viewed or their list can be downloaded from the following website https://pitgroup.org/amyloid .

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Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

Cited by 20 publications

References 38 publications

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

EmrE dimerization depends on membrane environment

PDB_Amyloid: an extended live amyloid structure list from the PDB

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