Work Description

Title: Negative Cooperativity Biochemical Data (Zlotnick Lab)

Open Access Deposited
http://creativecommons.org/licenses/by/4.0/
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Abstract
  • Hepatitis B Virus (HBV) capsid protein plays a role in almost every step of viral replication, from self-assembly to genome release. This makes capsid protein an attractive target for antiviral development. Capsid Assembly Modulators (CAMs) are direct-acting antivirals that bind capsid protein, leading to empty or aberrant complexes instead of an infectious virus. CAMs can also disrupt the capsid, preventing new infection. To understand CAM activity we examined the energetics of CAM-capsid interaction. We observed negative cooperativity of CAM binding kinetics and thermodynamics. These correlate with large changes in capsid architecture that presage capsid rupture. We propose that CAMs induce an accumulated strain on the capsid. Understanding the interplay of forces and architecture will help guide the development of new CAMs.
Methodology
  • 1. Fluorescence Polarization for kinetic measurements (Neo2 plate reader; BioTek, Winooski, VT). An other use of plate reader includes an excitation and emission spectrum. 2. Fluorimeter for kinetic measurements and light scattering (Photon Technology International fluorimeter accessorized with a rapid mixing stopped-flow unit - RX2000 Applied Photophysics). 3. Size-exclusion chromatography for equilibrium/absorbance measurements (Superose6 Increase 10/300 column; Cytiva). Samples were plunged through a Shimadzu HPLC equipped with a diode array absorbance detector (SPD-M20A) for absorbance measurements as samples eluted from the column. 4. Ultrafugation and UV-VIS spectrophotometer for equilibrium measurements. Ultrafugation with a Beckman Coulter, Optima MAX-XP in a TLA 100 rotor at 150,000 g x 22°C x 60 min. Absorance measured with an Agilent 8453 UV-Visible spectrophotometer. Other absorbance measurements, without an ultrafuge or chomatography step, were also taken on this spectrophotometer. 5. Total internal reflection fluorescennce microscopy for single-particle quantitation. Images were processed and analyzed using Andor Solis, Microsoft Excel, ImageJ and IgorPro software. 6. Transmission Electron Microscopy (JEOL 1010) at 20,000x zoom for capsid micrographs. 7. Mass Photometer (Refeyn TwoMP). All data processed in Microsoft Excel. Chemical structures drawn in ChemDraw.
Description
  • All data was processed in Microsoft Excel. An exception is the TIRF microscopy data, which led to Microsoft Excel for final computations and building the histogram. Across all data, measurements were taken from either the absorbance or the fluorescence of a fluorescent molecule (CAM-ALEXA488, 496/515 nm) and capsid protein (280 nm).
Creator
Depositor
Contact information
Funding agency
  • National Institutes of Health (NIH)
Keyword
Citations to related material
Publisher
Resource type
Last modified
  • 10/14/2025
Language
Doi
License
To Cite this Work:
Valkner, C., Zlotnick, A. Negative Cooperativity Biochemical Data (Zlotnick Lab) [Data set]. Indiana University - DataCORE. https://doi.org/10.5967/6q49-xt78

Files (Count: 2; Size: 33.5 MB)

Thumbnail Title Original Upload Last Modified File Size Access Actions
Figure_1___2_data.xlsx Figure_1___2_data.xlsx 2025-10-14 2025-10-14 20.2 MB Open Access
Suppl_Fig_Data.xlsx Suppl_Fig_Data.xlsx 2025-10-14 2025-10-14 13.3 MB Open Access

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