Mass spectrometry and non-covalent protein-ligand complexes: Confirmation of binding sites and changes in tertiary structure

Shields, Sharon J.; Oyeyemi, Olayinka; Lightstone, Felice C.; Balhorn, Rod

doi:10.1016/s1044-0305(03)00129-6

Cited by 35 publications

(31 citation statements)

References 44 publications

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“…7,11,12 In the present study we have four adjacent Arg followed by a Lys and an Arg on the basic peptide and six adjacent acidic residue plus a phosphate or six adjacent acidic residue or just one phosphate on the acidic peptide. Hence the possibility, that at least some of the NCX formed by these two acidic peptides KVNpSEEEEEDA and KVNSEEEEEDA, could be involving not just two adjacent Arg (from the basic peptide), but multiple Arg interacting with not just two adjacent Glu or a phosphate, but multiple Glu and the phosphate when available.…”

Section: Discussionmentioning

confidence: 91%

Phosphate Stabilization of Intermolecular Interactions

et al. 2005

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Receptor heteromerization is an important phenomenon that results from the interaction of epitopes on two receptors. Previous studies have suggested the possibility of Dopamine D 2 -NMDA receptors' interaction. We believe that the interaction is through an acidic epitope of the NMDA NR 1 subunit (KVNSEEEEEDA) and a basic epitope of the D 2 third intracellular loop (VLRRRRRKRVN), which was shown to also interact with the Adenosine A 2 A receptor. In previous work we highlighted the role of certain amino acid residues, mainly two or more adjacent arginine on one peptide and two or more adjacent glutamate, or aspartate, or a phosphorylated residue on the other in the formation of noncovalent complexes (NCX) between epitopes. In the present work, we use the phosphorylated (KVNSpEEEEEDA), non-phosphorylated (KVNSEEEEEDA) and modified (KVNpSAAAAAAA) forms of the NMDA epitope that possibly interact with the D 2 epitope to investigate the gas-phase stability of the NCXs as a function of the nominal energy given to the NCX ion as it enters the collision cell. In addition to theoretical calculations, the experimental data was used to calculate the stability of each electrostatic complex versus that of the dimer of KVNSpEEEEEDA. Our results demonstrate the importance of the phosphate group in stabilizing molecular interactions and that appreciably higher collision energies are required to completely dissociate any of the three different NCX ions that are formed through electrostatic interaction in comparison to the energy required to dissociate the KVNpSEEEEEDA dimer ion, which is mainly kept together by hydrogen bonding. This study emphasizes ionic bonds stability and their importance to protein structure as their potent electrostatic attractions can in the gas phase surpass the strength of covalent bonds.

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Section: Discussionmentioning

confidence: 91%

Phosphate Stabilization of Intermolecular Interactions

et al. 2005

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“…Changes in surface topology due to ligand binding can be inferred by comparing the relative ion abundances of peptides released from proteolysis of protein and protein-ligand complexes. A decrease in ion abundance suggests that the ligand obstructs the access of the protease to specific sites on the protein, identifying ligand-binding site(s) (Shields et al, 2003).…”

Section: Risedronate Binds To Cra In Vitromentioning

confidence: 99%

“…Trypsin-mediated proteolysis of Cra and Cra-ligand was carried out in a 300 : 1 protein : enzyme (wt/wt) ratio, according to the method of Shields et al (2003). Cra (4 mM) and Cra : ligand mixtures (4 : 40 mM) in 10 mM ammonium acetate (pH 7.7) were incubated at room temperature for 15 min before being digested simultaneously.…”

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confidence: 99%

Evaluation of risedronate as an antibiofilm agent

Reshamwala

Mamidipally

Pissurlenkar

et al. 2016

Journal of Medical Microbiology

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Escherichia coli cra null mutants have been reported in the literature to be impaired in biofilm formation. To develop E. coli biofilm-inhibiting agents for prevention and control of adherent behaviour, analogues of a natural Cra ligand, fructose-1,6-bisphosphate, were identified based on two-dimensional similarity to the natural ligand. Of the analogues identified, those belonging to the bisphosphonate class of drug molecules were selected for study, as these are approved for clinical use in humans and their safety has been established. Computational and in vitro studies with purified Cra protein showed that risedronate sodium interacted with residues in the fructose-1,6-bisphosphate-binding site. Using a quantitative biofilm assay, risedronate sodium, at a concentration of 300-400 mM, was found to decrease E. coli and Salmonella pullorum biofilm formation by .60 %. Risedronate drastically reduced the adherence of E. coli cells to a rubber Foley urinary catheter, demonstrating its utility in preventing the formation of biofilm communities on medical implant surfaces. The use of risedronate, either alone or in combination with other agents, to prevent the formation of biofilms on surfaces is a novel finding that can easily be translated into practical applications.

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“…30,31) In addition, susceptibility to proteolysis has been shown to be a sensitive method for detecting subtle conformation changes in proteins. 32,33) In this study, we examined the effect of putrescine on the conformation and activity of AdoMetDC purified from rat prostate using MALDI-TOF MS, and the susceptibility of AdoMetDC to trypsin following treatment with iodoacetic acid (IAA). We also examined the correlation between the presence of putrescine and stimulation of AdoMetDC activity and incorporation of IAA.…”

mentioning

confidence: 99%

Conformational Stabilization of Rat S-Adenosylmethionine Decarboxylase by Putrescine

Wada

Shirahata

2010

Biological & Pharmaceutical Bulletin

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The activity and processing of mammalian S-adenosylmethionine decarboxylase (AdoMetDC) is stimulated by putrescine. To obtain new insights into the mechanism through which putrescine stimulates AdoMetDC, we investigated conformational changes in rat prostate AdoMetDC in the presence or absence of putrescine. We examined the reactivity of purified rat prostate AdoMetDC to the SH-reagent iodoacetic acid (IAA) and its susceptibility to proteolysis in the presence or absence of putrescine using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The activity of AdoMetDC treated with IAA in the absence of putrescine was reduced, but about 80% of its activity remained after treatment with IAA in the presence of putrescine. In the presence of putrescine, IAA incorporation was 1.9 mol IAA/mol of AdoMetDC a-subunit, while there was no incorporation of IAA in the b-subunit of AdoMetDC. In the absence of putrescine, 5.0 mol of IAA/mol of a-subunit and 0.9 mol of IAA/mol of b-subunit were incorporated. Only Cys292 and Cys310 were carboxymethylated by IAA in the presence of putrescine. In contrast, in the absence of putrescine all cysteines were carboxymethylated by IAA. In addition, putrescine slowed the rate of AdoMetDC degradation by trypsin. These results demonstrate that the conformation of AdoMetDC purified from rat prostate is stabilized by putrescine.

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Mass spectrometry and non-covalent protein-ligand complexes: Confirmation of binding sites and changes in tertiary structure

Cited by 35 publications

References 44 publications

Phosphate Stabilization of Intermolecular Interactions

Phosphate Stabilization of Intermolecular Interactions

Evaluation of risedronate as an antibiofilm agent

Conformational Stabilization of Rat S-Adenosylmethionine Decarboxylase by Putrescine

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