Studies of protein-drug interactions by mass spectrometry

Pao Yue-kong Library Electronic Theses Database

Studies of protein-drug interactions by mass spectrometry


Author: So, Pui-kin
Title: Studies of protein-drug interactions by mass spectrometry
Degree: Ph.D.
Year: 2008
Subject: Hong Kong Polytechnic University -- Dissertations.
Mass spectrometry.
Electrospray ionization mass spectrometry.
Protein drugs.
Department: Dept. of Applied Biology and Chemical Technology
Pages: xx, 170, [11] p. : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record:
Abstract: b-Lactamase, a bacterial enzyme that hydrolyzes b-lactam antibiotics, has been recently modified and labeled with different fluorophores to act as fluorescence biosensors for detecting residual b-lactam antibiotics in food. E166Cf was constructed by replacing the glutamate residue on the Q-loop of Class A b-lactamases at position 166 with cysteine to produce the E166C mutant, to which a fluorophore, fluorescein-5-maleimide, is attached. A second biosensor E166Cb was constructed by labeling with the fluorophore badan. These two biosensors were found to be able to emit enhanced and intense fluorescent signals upon specific binding to b-lactam antibiotics, enabling them to detect antibiotics like penicillin G, cefuroxime and cefotaxime down to the nanomolar level (10 -9 M). To understand the biosensing mechanism of the b-lactamase-based biosensors, the kinetics and structural basis of the binding reactions between the biosensors and b-lactam antibiotics were investigated by electrospray ionization mass spectrometry (ESI-MS). The identities of E166C and its fluorophore-labeled mutants were confirmed by ESI-MS. Complementary time-dependent mass spectrometric and fluorometric studies show that, in general, the time-resolved fluorescence profile correlate well with the concentration-time profile of the covalently bound enzyme-substrate complex (ES*, where E is E166Cf or E166Cb, and S is the substrate b-lactam antibiotic) monitored by ESI-MS. This observation demonstrates unambiguously that the fluorescence emission enhancement is due to specific substrate binding of a b-lactam type antibiotic. Compared to the wild-type b-lactamases, detailed kinetics studies revealed that the hydrolytic (deacylation) dissociation rate (k3 values) of the E166C, E166Cf and E166Cb mutants are much reduced to the order of 10-4 s-1, and are ~ 5 orders of magnitude smaller than the rate of formation of ES* (k2 values in the order of 101 s-1). These kinetic properties ensure that ES* are formed at high concentrations at steady state, but dissociate very slowly thereafter. Consequently, a steady and intense fluorescent signal can be easily monitored over a reasonable analysis time period (say 1,000 seconds) in practical applications. On the other hand, the specific binding efficiencies towards b-lactam antibiotics were found to have maintained and even enhanced for the E166C, E166Cf and E166Cb mutants. The overall binding efficiency, as indicated by the ratio of kinetic parameters k2/Kd, is not significantly impaired by the introduction of fluorescein-5-maleimide at the C166 position of the Q-loop of the b-lactamases, which is located near the active binding site. This is attributed to that the flexible nature of the Q -loop and fluorophore-induced increase in flexibility of the active site binding pocket, thereby relieving the steric crowding effect exerted by the fluorophore. The effect of using a bulky fluorescein-5-maleimide versus a smaller but hydrophobic badan fluorophore in the biosensor were investigated. The fluorescence characteristics of E166Cf and E166Cb were found to be different. Specifically, the incorporation of badan was found to enhance the overall binding efficiency more significantly by ~10-folds. This surprising result might be due to the highly hydrophobic nature of badan, which tends to repel/displace the surrounding dense water clusters which serve to stabilize the structural integrity of the active site binding pocket. With the loss/lowering of the stabilizing effects offered by these water molecules, the flexibility of the active site would be further enhanced, and therefore the steric blocking effect imposed by the fluorophore could be alleviated to a greater extent. Mass spectrometric hydrogen-deuterium (H/D) exchange studies showed that for E166Cf and E166Cb, the H/D exchange levels of two peptide segments near the active site are higher than those of free E166C, indicating that the fluorophore may have induced local dynamic changes to the active site region. However, upon substrate binding, the H/D exchange levels of these two segments of E166Cf and E166Cb decrease and become similar to that of E166C, indicating that the dynamic changes mediated by the fluorophore are nullified. Based on these observations, a "spatial displacement" mechanism was proposed. The fluorophore (both fluorescein-5-maleimide and badan) may initially be oriented towards and close to the active binding site, and induce destabilizing effect to this confined region by displacing the "structural glue" water molecules and disrupting some noncovalent interactions involved in maintaining the structural integrity of the active binding pocket. Upon substrate binding, the fluorophore is displaced away from the active site in order to avoid the spatial clash with the incoming substrate, thus the destabilizing effects initially exerted by the fluorophore to this region are also withdrawn. This spatial displacement event is the major cause for changes in local environment surrounding the fluorophore, i.e. solvent polarity and accessibility, thereby changing the fluorescence emission properties of the fluorophore. This proposed mechanism is found to be consistent with the results of molecular modeling and preliminary X-ray crystallographic studies. Due to discrepancies in reported results obtained by ESI-MS under acidic, denaturating conditions and other physical techniques such as X-ray crystallography and UV spectroscopy, the inhibition mechanism of tazobactam (MW=300 Da) towards b-lactamases was re-investigated by ESI-MS but under near physiological ( pH 7 ) conditions. Unlike previous ESI-MS studies, a covalently bound enzyme-inhibitor complex (E-I complex) with a relative molar mass of [M + 300] Da was observed (M is the average molecular mass of the enzyme protein), which is consistent with the formation of a trans-enamine species as suggested by X-ray crystallography and UV spectroscopic methods. In addition, our results show that, for the first time, the E-I complex formed from the Staphylococcus aureus PC-1 b-lactamase and tazobactam dissociates further to form an inactive dehydrated enzyme. Based on the results obtained by protease digestion and tandem mass spectrometry, this dehydrated enzyme is proposed to be an alkene-like species formed from dissociation of the trans-enamine species. For this inhibition mechanism, the role of the inhibitor is initial binding to the active site of the enzyme, followed by triggering of a chemical reaction (or reactions) that result in the formation of an inactivated form of b-lactamase.

Files in this item

Files Size Format
b22762607.pdf 3.859Mb PDF
Copyright Undertaking
As a bona fide Library user, I declare that:
  1. I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
  2. I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
  3. I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.


Quick Search


More Information