Folding of phage P22 coat protein monomers: kinetic and thermodynamic properties

Anderson, Eric H.; Teschke, Carolyn M.

doi:10.1016/s0042-6822(03)00240-x

Cited by 25 publications

(49 citation statements)

References 67 publications

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“…The most surprising result came from the kinetic experiments monitored by CD. Both the unfolding and refolding reactions of the tsf coat proteins are too fast to be monitored by manual mixing experiments (with a dead time of ϳ5-7 s), whereas WT coat protein had readily observable kinetics for both reactions (21). From our experiments, we concluded that the domain of secondary structure, which is monitored by CD, is "flickering" in and out of its native state and populating an intermediate.…”

mentioning

confidence: 82%

“…Unfolded and Refolded Coat Protein Monomers-To obtain unfolded coat protein, empty procapsid shells were incubated in 6.75 M urea for 30 min at room temperature, which dissociates and denatures the subunits to monomers (21,22,25). Refolded coat protein monomers were formed by first denaturing empty procapsid shells in 6.75 M urea as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Unfolding and Refolding to Equilibrium-Samples for urea equilibrium titration curves were made using a Hamilton Microlab 500 titrator as described previously (21,22). Approximately 70 samples were used for each technique to define the equilibrium curves.…”

Section: Methodsmentioning

confidence: 99%

“…Our initial in vitro investigations of WT coat protein and tsf coat proteins determined how the tsf amino acid substitutions affect the folding and assembly of coat protein (21,22). We found that coat protein has two folding domains defined by spectroscopic probes: a domain of secondary structure primarily monitored by circular dichroism (CD) and a hydrophobic domain with a tryptophan pocket, which can be monitored by tryptophan fluorescence.…”

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confidence: 99%

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A Concerted Mechanism for the Suppression of a Folding Defect through Interactions with Chaperones

Doyle

Anderson

Parent

et al. 2004

Journal of Biological Chemistry

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Specific amino acid substitutions confer a temperature-sensitive-folding (tsf ) phenotype to bacteriophage P22 coat protein. Additional amino acid substitutions, called suppressor substitutions (su), relieve the tsf phenotype. These su substitutions are proposed to increase the efficiency of procapsid assembly, favoring correct folding over improper aggregation. Our recent studies indicate that the molecular chaperones GroEL/ES are more effectively recruited in vivo for the folding of tsf:su coat proteins than their tsf parents. Here, the tsf:su coat proteins are studied with in vitro equilibrium and kinetic techniques to establish a molecular basis for suppression. The tsf:su coat proteins were monomeric, as determined by velocity sedimentation analytical ultracentrifugation. The stability of the tsf:su coat proteins was ascertained by equilibrium urea titrations, which were best described by a three-state folding model, N N I N U. The tsf:su coat proteins either had stabilized native or intermediate states as compared with their tsf coat protein parents. The kinetics of the I N U transition showed a decrease in the rate of unfolding and a small increase in the rate of refolding, thereby increasing the population of the intermediate state. The increased intermediate population may be the reason the tsf:su coat proteins are aggregation-prone and likely enhances GroEL-ES interactions. The N f I unfolding rate was slower for the tsf:su proteins than their tsf coat parents, resulting in an increase in the native state population, which may allow more competent interactions with scaffolding protein, an assembly chaperone. Thus, the suppressor substitution likely improves folding in vivo through increased efficiency of coat protein-chaperone interactions.The processes of protein folding and assembly are driven by the primary amino acid sequence (1, 2). Changes in this sequence, such as amino acid substitutions or deletions, can lead to protein misfolding and aggregation (3). These protein folding problems have been linked with serious human diseases (4 -6). For example, a change in the amino acid sequence of the cystic fibrosis transmembrane receptor, most commonly ⌬F508, causes misfolding and aggregation leading to cystic fibrosis (5,7,8). Osteogenesis imperfecta is caused by protein misfolding and is induced by many different amino acid substitutions that weaken the collagen fibrils (4, 5, 9). The seriousness of these diseases highlights the significance of specific amino acids in the processes of protein folding and aggregation.Our model system for studying the effects of amino acid substitutions on folding and assembly is coat protein of P22, a double stand DNA bacteriophage of Salmonella typhimurium. P22 coat protein is a 47-kDa polypeptide comprising 429 amino acids (10, 11). During assembly, 420 coat protein monomers and 150 -300 molecules of scaffolding protein, an assembly chaperone, form a spherical procapsid into which DNA is packaged to form a phage (12-16). Single amino acid substitutions in the coat pro...

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mentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Concerted Mechanism for the Suppression of a Folding Defect through Interactions with Chaperones

Doyle

Anderson

Parent

et al. 2004

Journal of Biological Chemistry

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“…WT, tsf, and tsf :su coat proteins fold to their native conformation via a 3-state equilibrium folding model, unfolded ⇔ intermediate ⇔ native (U ⇔ I ⇔ N) (Teschke and King 1993;Anderson and Teschke 2003;Doyle et al 2003Doyle et al , 2004. A late-folding intermediate has been identified to interact with GroEL (de Beus et al 2000).…”

Section: Chaperone Roles In Protein Folding and In Capsid Assemblymentioning

confidence: 99%

GroEL/S substrate specificity based on substrate unfolding propensity

Parent

Teschke

2007

Cell Stress Chaper

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Phage P22 wild-type (WT) coat protein does not require GroEL/S to fold but temperature-sensitive-folding (tsf ) coat proteins need the chaperone complex for correct folding. WT coat protein and all variants absolutely require P22 scaffolding protein, an assembly chaperone, to assemble into precursor structures termed procapsids. Previously, we showed that a global suppressor (su) substitution, T166I, which rescues several tsf coat protein variants, functioned by inducing GroEL/S. This led to an increased formation of tsf :T166I coat protein:GroEL complexes compared with the tsf parents. The increased concentration of complexes resulted in more assembly-competent coat proteins because of a shift in the chaperone-driven kinetic partitioning between aggregation-prone intermediates toward correct folding and assembly. We have now investigated the folding and assembly of coat protein variants that carry a different global su substitution, F170L. By monitoring levels of phage production in the presence of a dysfunctional GroEL we found that tsf :F170L proteins demonstrate a less stringent requirement for GroEL. Tsf :F170L proteins also did not cause induction of the chaperones. Circular dichroism and tryptophan fluorescence indicate that the native state of the tsf : F170L coat proteins is restored to WT-like values. In addition, native acrylamide gel electrophoresis shows a stabilized native state for tsf :F170L coat proteins. The F170L su substitution also increases procapsid production compared with their tsf parents. We propose that the F170L su substitution has a decreased requirement for the chaperones GroEL and GroES as a result of restoring the tsf coat proteins to a WT-like state. Our data also suggest that GroEL/S can be induced by increasing the population of unfolding intermediates.

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Salmonella Phages and Prophages: Genomics, Taxonomy, and Applied Aspects

Switt

Sulakvelidze

Wiedmann

et al. 2014

Methods in Molecular Biology

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Since this book was originally published in 2007 there has been a significant increase in the number of Salmonella bacteriophages, particularly lytic virus, and Salmonella strains which have been fully sequenced. In addition, new insights into phage taxonomy have resulted in new phage genera, some of which have been recognized by the International Committee of Taxonomy of Viruses (ICTV). The properties of each of these genera are discussed, along with the role of phage as agents of genetic exchange, as therapeutic agents, and their involvement in phage typing.

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Folding of phage P22 coat protein monomers: kinetic and thermodynamic properties

Cited by 25 publications

References 67 publications

A Concerted Mechanism for the Suppression of a Folding Defect through Interactions with Chaperones

A Concerted Mechanism for the Suppression of a Folding Defect through Interactions with Chaperones

GroEL/S substrate specificity based on substrate unfolding propensity

Salmonella Phages and Prophages: Genomics, Taxonomy, and Applied Aspects

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