Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes

Hanes, Melinda S.; Ratcliff, Kathleen; Marqusee, Susan; Handel, Tracy M.

doi:10.1002/pro.467

Cited by 7 publications

(6 citation statements)

References 11 publications

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“…Structures were visualized with PyMOL (46). TEM-1 β-lactamase with the M182T stabilizing mutation and all cysteine variants of this background sequence were purified from the periplasmic fraction of BL21(DE3) cells as described in previous work (47) and SI Materials and Methods. We measured enzyme activities following previous work (48), as described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Bowman

Bolin

Hart

et al. 2015

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

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The discovery of drug-like molecules that bind pockets in proteins that are not present in crystallographic structures yet exert allosteric control over activity has generated great interest in designing pharmaceuticals that exploit allosteric effects. However, there have only been a small number of successes, so the therapeutic potential of these pockets-called hidden allosteric sites-remains unclear. One challenge for assessing their utility is that rational drug design approaches require foreknowledge of the target site, but most hidden allosteric sites are only discovered when a small molecule is found to stabilize them. We present a means of decoupling the identification of hidden allosteric sites from the discovery of drugs that bind them by drawing on new developments in Markov state modeling that provide unprecedented access to microsecond-to millisecond-timescale fluctuations of a protein's structure. Visualizing these fluctuations allows us to identify potential hidden allosteric sites, which we then test via thiol labeling experiments. Application of these methods reveals multiple hidden allosteric sites in an important antibiotic target-TEM-1 β-lactamase. This result supports the hypothesis that there are many as yet undiscovered hidden allosteric sites and suggests our methodology can identify such sites, providing a starting point for future drug design efforts. More generally, our results demonstrate the power of using Markov state models to guide experiments.thiol labeling | antibiotic resistance | molecular dynamics

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Section: Methodsmentioning

confidence: 99%

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Bowman

Bolin

Hart

et al. 2015

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

Self Cite

182

232

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“…3,10 Moreover, under certain conditions, its relationship with protein stability has been utilized to determine physical parameters such as free energy of unfolding and binding constants of ligands using pulse proteolysis. [11][12][13] Also, the linkage of proteolytic resistance with protein stability has been extensively utilized to evolve stable variants of a numbers of proteins and peptides using protein stability increase by directed evolution (Proside). [14][15][16][17][18] In addition, proteolytic resistance has also been exploited for protein purification.…”

Section: Introductionmentioning

confidence: 99%

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

et al. 2012

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Improvement in protein thermostability was often found to be associated with increase in its proteolytic resistance as revealed by comparative studies of homologous proteins from extremophiles or mutational studies. Structural elements of protein responsible for this association are not firmly established although loops are implicated indirectly due to their structural role in protein stability. To get a better insight, a detailed study of protein wide mutants and their influence on stability and proteolytic resistance would be helpful. To generate such a data set, a model protein, Bacillus subtilis lipase was subjected to loop scanning site-saturation mutagenesis on 86 positions spanning all loops including termini. Upon screening of~16,000 clones, 17 single mutants with improved thermostability were identified with increment in apparent melting temperature (Tm app ) by 1-6°C resulting in an increase in free energy of unfolding (DG unf ) by 0.04-1.16 kcal/mol. Proteolytic resistance of all single mutants upon incubation with nonspecific protease, Subtilisin A, was determined. Upon comparison, post-proteolysis residual activities as well as kinetics of proteolysis of mutants showed excellent correlation with DG unf , (r > 0.9), suggesting that proteolysis was strongly correlated with the global stability of this protein. This significant correlation in this set, with least possible sequence changes (single aa substitution), while covering >60% of protein surface strongly argues for the covariance of these two variables. Compared to studies from extremophiles, with large sequence heterogeneity, the observed correlation in such a narrow sequence space (DDG unf 5 1.57 kcal 21 ) justifies the robustness of this relation.

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“…The well-established affinity between TEM-1 β-lactamase and BLIP has been the focus of a number of recent studies to design new tools. Using this interaction, Yuan et al (2009) proposed a genetic screen for BLIP function, Hanes et al (2010) developed a simple and robust pulse proteolysis method focusing on binding affinity, and Fryszczyn et al (2011) offered an improvement in phage display technology. FRET-based techniques that utilize the TEM-1 β-lactamase:BLIP interaction were used to measure binding in the cytoplasm of HeLa cells to test protein association in a crowded environment, and for high-throughput analysis of protein-protein interactions (Khait and Schreiber, 2012;Phillip et al, 2012aPhillip et al, , 2012b.…”

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confidence: 99%

Investigation of the in vivo interaction between β-lactamaseand its inhibitor protein

Gökgöz

Yalaz

Avci³

et al. 2015

Turk J Biol

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The affinity of β-lactamase inhibitory protein (BLIP) for TEM-1 β-lactamase has raised hopes in the challenge of proteinbased inhibitor discovery for β-lactamase-mediated antibiotic resistance. Currently, the effect of the formation of the β-lactamase:BLIP complex in vivo in β-lactam resistant bacteria is an open question. The scarcity of information to the extent to which BLIP can impair β-lactamase activity inside cells has urged us to assess the in vivo efficacy of BLIP as a potent β-lactamase inhibitor. To this end, β-lactamase and BLIP were coexpressed in Escherichia coli. Simultaneous expression of β-lactamase and BLIP and the formation of the TEM-1 β-lactamase:BLIP complex in the periplasmic space of E. coli were verified by electrophoretic and Western blot techniques. Growth profiles of the cells expressing both β-lactamase and its protein inhibitor, complemented with β-lactamase activity measurements, suggested that BLIP synthesis retarded cell growth and reduced β-lactamase activity. Although co-expression of β-lactamase and its protein inhibitor did not completely impair cell growth, the specificity of BLIP enabled it to bind β-lactamase in the bacterial periplasm, regardless of the crowding components.

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Protein–protein binding affinities by pulse proteolysis: Application to TEM‐1/BLIP protein complexes

Cited by 7 publications

References 11 publications

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Discovery of multiple hidden allosteric sites by combining Markov state models and experiments

Probing protein stability and proteolytic resistance by loop scanning: A comprehensive mutational analysis

Investigation of the in vivo interaction between β-lactamaseand its inhibitor protein

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