Protein Quaternary Structures in Solution are a Mixture of Multiple forms

Marciano, Shir; Dey, Debabrata; Listov, Dina; Fleishman, Sarel J.; Sonn-Segev, Adar; Mertens, Haydyn D. T.; Büsch, Florian; Kim, Yong-Seok; Harvey, Sophie R.; Wysocki, Vicki H.; Schreiber, Gideon

doi:10.1101/2022.03.30.486392

Cited by 3 publications

(6 citation statements)

References 79 publications

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“…Similar to Aerogen-nebulized IFN-α2, PARI-nebulized BSA also presents increasing recovery along with input concentration (Figure A). In contrast, the T m increased only slightly at higher concentrations (Figure B), possibly due to the tendency of BSA to dimerize . Gelatin alone did not show significant temperature-dependent signals in the Tycho NT6 (Figure C,D).…”

Section: Resultsmentioning

confidence: 92%

“…The T m increased exponentially from 58 to 63 °C with increasing input protein concentrations (Figure B). This is attributed to the concentration-dependent dimerization of IFN-α2, which increases its thermostability . Next, we repeated the same experiments using RBD-62 (Figure C,D) with 50–400 μg/mL input concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…This is attributed to the concentration-dependent dimerization of IFN-α2, which increases its thermostability. 29 Next, we repeated the same experiments using RBD-62 ( Figure 2 C,D) with 50–400 μg/mL input concentrations. The fraction recovery displays the same increasing behavior as that of IFN-α2.…”

Section: Resultsmentioning

confidence: 99%

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Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19

Marton

Harari

et al. 2022

ACS Biomater. Sci. Eng.

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Delivering medication to the lungs via nebulization of pharmaceuticals is a noninvasive and efficient therapy route, particularly for respiratory diseases. The recent worldwide severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic urges the development of such therapies as an effective alternative to vaccines. The main difficulties in using inhalation therapy are the development of effective medicine and methods to stabilize the biological molecules and transfer them to the lungs efficiently following nebulization. We have developed a high-affinity angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD-62) that can be used as a medication to inhibit infection with SARS-CoV-2 and its variants. In this study, we established a nebulization protocol for drug delivery by inhalation using two commercial vibrating mesh (VM) nebulizers (Aerogen Solo and PARI eFlow) that generate similar mist size distribution in a size range that allows efficient deposition in the small respiratory airway. In a series of experiments, we show the high activity of RBD-62, interferon-α2 (IFN-α2), and other proteins following nebulization. The addition of gelatin significantly stabilizes the proteins and enhances the fractions of active proteins after nebulization, minimizing the medication dosage. Furthermore, hamster inhalation experiments verified the feasibility of the protocol in pulmonary drug delivery. In short, the gelatin-modified RBD-62 formulation in coordination with VM nebulizer can be used as a therapy to cure SARS-CoV-2.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19

Marton

Harari

et al. 2022

ACS Biomater. Sci. Eng.

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“…These proteins include triose phosphate isomerase (8TIM), streptavidin (1SWB), neutravidin (1AVE), pyruvate kinase (1AQF), concanavalin A (1JBC), transthyretin (5HJG), D-sialic acid aldolase (6ALD), hemoglobin (1GZX), tryptophan synthetase (1WBJ), cholera toxin B (1FGB), C-reactive protein (1GNH), serum amyloid P (1SAC), beta-lactoglobulin (6QI6), lysozyme (4R0F), and enolase (1E9I). A few additional proteins were also included in the data set: , IspD (1VGT), Can (1 T75), DeoC (1KTN), Upp (2EHJ), and HFq (1HK9). In all cases, experiments were performed on a Waters Synapt G2 or G2s mass spectrometer, operated in mobility mode.…”

Section: Methodsmentioning

confidence: 99%

Simulation of Energy-Resolved Mass Spectrometry Distributions from Surface-Induced Dissociation

et al. 2022

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Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from the structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (R 2 = 0.71). Furthermore, based on this trend, we used native crystal structures to predict ERMS. The majority of predictions resulted in good agreement, and the average root-mean-square error was 0.20 for the 20 complexes in our data set. We also show that if additional information on cleavage as a function of collision energy could be obtained, the accuracy of predictions improved further. Finally, we demonstrated that ERMS prediction results were better for the native than for inaccurate models in 17/20 cases. An application to run this simulation has been developed in Rosetta, which is freely available for use.

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“…These proteins include triose phosphate isomerase (8TIM), streptavidin (1SWB), neutravidin (1AVE), pyruvate kinase (1AQF), concanavalin A (1JBC), transthyretin (5HJG), D-sialic acid aldolase (6ALD), hemoglobin (1GZX), tryptophan synthetase (1WBJ), cholera toxin B (1FGB), C-reactive protein (1GNH), and serum amyloid P (1SAC). A few additional proteins were also included in the dataset (41,42): IspD (1VGT), Can (1T75), DeoC (1KTN), Upp (2EHJ), and HFq (1HK9). In all cases experiments were performed on a Waters Synapt G2 or G2s mass spectrometer, operated in mobility mode.…”

Section: Benchmark Setmentioning

confidence: 99%

Simulation of energy-resolved mass spectrometry distributions from surface-induced dissociation

Seffernick

Turzo

Harvey

et al. 2022

Preprint

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Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved tandem MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (R^2 = 0.71). Furthermore, based on this trend, we used native crystal structures to predict ERMS. The majority of predictions resulted in good agreement, and the average root-mean-square error (RMSE) was 0.20 for the 20 complexes in our dataset. We also show that if additional information on cleavage as a function of collision energy could be obtained, the accuracy of predictions improved further. Finally, we demonstrated that ERMS prediction results were better for the native than for inaccurate models in 17/20 cases. An application to run this simulation has been developed in Rosetta, which is freely available for use.

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Protein Quaternary Structures in Solution are a Mixture of Multiple forms

Cited by 3 publications

References 79 publications

Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19

Gelatin Stabilizes Nebulized Proteins in Pulmonary Drug Delivery against COVID-19

Simulation of Energy-Resolved Mass Spectrometry Distributions from Surface-Induced Dissociation

Simulation of energy-resolved mass spectrometry distributions from surface-induced dissociation

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