A New Ultrasensitive Scanning Calorimeter

Plotnikov, Valerian; Brandts, J.Michael; Lin, Lung‐Nan; Brandts, John F.

doi:10.1006/abio.1997.2236

Cited by 166 publications

(100 citation statements)

References 10 publications

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“…The area of the peak observed in the DSC scan represents the enthalpy change for the heat-induced process. Several advancements in differential scanning calorimeters over the past few decades provided enhanced sensitivity and signal detection (21)(22)(23). Such developments allow a broader range of applications and interrogation of more diverse experimental systems.…”

Section: Resultsmentioning

confidence: 99%

“…Plotting the raw Dp-versus-time data shows that increasing the scan rate from 90 to 200°C/h shortens the duration of the DNA ejection peak from 500 s to 150 s, which noticeably increases the raw calorimetric signal intensity. This effect (along with technical enhancements in newer-generation calorimeters [21][22][23]) may explain why previous DSC studies on phage did not capture the comparatively smaller enthalpy of genome release relative to protein denaturation and DNA melting (24).…”

Section: Resultsmentioning

confidence: 99%

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Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Bauer

Huffman

et al. 2015

J Virol

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We have recently shown in both herpesviruses and phages that packaged viral DNA creates a pressure of tens of atmospheres pushing against the interior capsid wall. For the first time, using differential scanning microcalorimetry, we directly measured the energy powering the release of pressurized DNA from the capsid. Furthermore, using a new calorimetric assay to accurately determine the temperature inducing DNA release, we found a direct influence of internal DNA pressure on the stability of the viral particle. We show that the balance of forces between the DNA pressure and capsid strength, required for DNA retention between rounds of infection, is conserved between evolutionarily diverse bacterial viruses (phages and P22), as well as a eukaryotic virus, human herpes simplex 1 (HSV-1). Our data also suggest that the portal vertex in these viruses is the weakest point in the overall capsid structure and presents the Achilles heel of the virus's stability. Comparison between these viral systems shows that viruses with higher DNA packing density (resulting in higher capsid pressure) have inherently stronger capsid structures, preventing spontaneous genome release prior to infection. This force balance is of key importance for viral survival and replication. Investigating the ways to disrupt this balance can lead to development of new mutation-resistant antivirals. IMPORTANCEA virus can generally be described as a nucleic acid genome contained within a protective protein shell, called the capsid. For many double-stranded DNA viruses, confinement of the large DNA molecule within the small protein capsid results in an energetically stressed DNA state exerting tens of atmospheres of pressures on the inner capsid wall. We show that stability of viral particles (which directly relates to infectivity) is strongly influenced by the state of the packaged genome. Using scanning calorimetry on a bacterial virus (phage ) as an experimental model system, we investigated the thermodynamics of genome release associated with destabilizing the viral particle. Furthermore, we compare the influence of tight genome confinement on the relative stability for diverse bacterial and eukaryotic viruses. These comparisons reveal an evolutionarily conserved force balance between the capsid stability and the density of the packaged genome.A virus can generally be described as a nucleic acid genome contained within a protective protein shell, termed the capsid. The viral replication cycle is composed of two distinct processes: delivery of the genetic material to the host (infection) and the subsequent production of new virions (replication). Between rounds of replication, the virion must be sufficiently stable to ensure that the packaged genome is retained within the capsid. Conversely, during infection it must be unstable enough to allow efficient genome release into the host cell. This balance between genome retention and release is described as viral metastability (1). Viral particles are therefore not inert structures and have not ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Bauer

Huffman

et al. 2015

J Virol

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“…Differential Scanning Calorimetry-A VP-DSC microcalorimeter (MicroCal, Northampton, MA) equipped with twin coin-shaped cells of 0.52-ml volume was used. Technical details and performance of the instrument have been described elsewhere (24). The protein was dialyzed for 18 h against the same batch of buffer that was used to establish the base line (25 mM potassium phosphate, pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

Thermosensor Action of GrpE

Grimshaw

Jelesarov

Siegenthaler

et al. 2003

Journal of Biological Chemistry

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Temperature directly controls functional properties of the DnaK/DnaJ/GrpE chaperone system. The rate of the high to low affinity conversion of DnaK shows a non-Arrhenius temperature dependence and above ϳ40°C even decreases. In the same temperature range, the ADP/ATP exchange factor GrpE undergoes an extensive, fully reversible thermal transition (Grimshaw, J. P. A., Jelesarov, I., Schö nfeld, H. J., and Christen, P. (2001) J. Biol. Chem. 276, 6098 -6104). To show that this transition underlies the thermal regulation of the chaperone system, we introduced an intersubunit disulfide bond into the paired long helices of the GrpE dimer. The transition was absent in disulfide-linked GrpE R40C but was restored by reduction. With disulfide-stabilized GrpE, the rate of ADP/ATP exchange and conversion of DnaK from its ADP-liganded high affinity R state to the ATP-liganded low affinity T state continuously increased with increasing temperature. With reduced GrpE R40C, the conversion became slower at temperatures >40°C, as observed with wild-type GrpE. Thus, the long helix pair in the GrpE dimer acts as a thermosensor that, by decreasing its ADP/ATP exchange activity, induces a shift of the DnaK⅐substrate complexes toward the high affinity R state and in this way adapts the DnaK/DnaJ/GrpE system to heat shock conditions. Cells respond to an increase in temperature by increased synthesis of heat shock proteins (Hsps).1 Molecular chaperone systems of the Hsp70 family prevent the formation of protein aggregates and facilitate the folding of nascent polypeptide chains and denatured proteins (for comprehensive reviews, see Refs. 1 and 2). DnaK, an Hsp70 homolog of Escherichia coli, binds peptides and segments of denatured proteins in extended conformation (3, 4) and cooperates with two cohort heat shock proteins: DnaJ, an Hsp40 homolog, and GrpE (5). The DnaK/ DnaJ/GrpE chaperone system has been extensively studied in vitro at ambient temperatures (6 -12). DnaK alternates between two states, the ATP-liganded low affinity T state with fast binding and release of the substrate and the ADP-liganded high affinity R state with slow kinetics. A substrate is first bound by T state DnaK, which is then converted to the high affinity R state through DnaJ-triggered hydrolysis of DnaKbound ATP. With the assistance of GrpE, which serves as an ADP/ATP exchange factor, DnaK is reconverted from the R state into the low affinity T state, releasing the substrate.Heat shock proteins, by definition, are induced by a heat shock, i.e. a transient increase in temperature enhances the expression level of chaperones and co-chaperones. The transcription of the genes of DnaK and its co-chaperones DnaJ and GrpE is controlled by the initiation factor 32 of RNA polymerase (for a recent review, see Ref. 13). Recently, we have investigated the direct effect of elevated temperatures on the isolated DnaK/DnaJ/GrpE chaperone system. GrpE, which is an elongated homodimer both in solution (14, 15) and in crystalline form (Fig. 1 and Ref. 16), has been found to und...

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“…Quantitative estimates of association for ligand binding to L99A T4 lysozyme were obtained by ITC using a Microcal VP-ITC calorimeter 53 operated at 10°C with a reference power of 10 μcal/s, a stirring speed of 300 rpm, and a data collection interval of 4 min per injection. An initial injection of 2 μl of ligand was followed by an additional 29 injections of 10 μl totaling 292 μl.…”

Section: Isothermal Titration Calorimetrymentioning

confidence: 99%

Predicting Absolute Ligand Binding Free Energies to a Simple Model Site

Mobley

Graves

Chodera

et al. 2007

Journal of Molecular Biology

311

501

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A central challenge in structure-based ligand design is the accurate prediction of binding free energies. Here we apply alchemical free energy calculations in explicit solvent to predict ligand binding in a model cavity in T4 lysozyme. Even in this simple site, there are challenges. We made systematic improvements, beginning with single poses from docking, then including multiple poses, additional protein conformational changes, and using an improved charge model. Computed absolute binding free energies had an RMS error of 1.9 kcal/mol relative to previously determined experimental values. In blind prospective tests, the methods correctly discriminated between several true ligands and decoys in a set of putative binders identified by docking. In these prospective tests, the RMS error in predicted binding free energies relative to those subsequently determined experimentally was only 0.6 kcal/mol. X-ray crystal structures of the new ligands bound in the cavity corresponded closely to predictions from the free energy calculations, but sometimes differed from those predicted by docking. Finally, we examined the impact of holding the protein rigid, as in docking, with a view to learning how approximations made in docking affect accuracy and how they may be improved.

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A New Ultrasensitive Scanning Calorimeter

Cited by 166 publications

References 10 publications

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Exploring the Balance between DNA Pressure and Capsid Stability in Herpesviruses and Phages

Thermosensor Action of GrpE

Predicting Absolute Ligand Binding Free Energies to a Simple Model Site

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