The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function

The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress \u3ci\u3ein Vivo\u3c/i\u3e Indicates That These Chaperones Protect a Wide Range of Cellular Functions

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function

In situ enrichment of ocean crust microbes on igneous minerals and glasses using an osmotic flow-through device

Author Posting. © American Geophysical Union, 2011. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geochemistry Geophysics Geosystems 12 (2011): Q06007, doi:10.1029/2010GC003424.The Integrated Ocean Drilling Program (IODP) Hole 1301A on the eastern flank of Juan de Fuca Ridge was used in the first long-term deployment of microbial enrichment flow cells using osmotically driven pumps in a subseafloor borehole. Three novel osmotically driven colonization systems with unidirectional flow were deployed in the borehole and incubated for 4 years to determine the microbial colonization preferences for 12 minerals and glasses present in igneous rocks. Following recovery of the colonization systems, we measured cell density on the minerals and glasses by fluorescent staining and direct counting and found some significant differences between mineral samples. We also determined the abundance of mesophilic and thermophilic culturable organotrophs grown on marine R2A medium and identified isolates by partial 16S or 18S rDNA sequencing. We found that nine distinct phylotypes of culturable mesophilic oligotrophs were present on the minerals and glasses and that eight of the nine can reduce nitrate and oxidize iron. Fe(II)-rich olivine minerals had the highest density of total countable cells and culturable organotrophic mesophiles, as well as the only culturable organotrophic thermophiles. These results suggest that olivine (a common igneous mineral) in seawater-recharged ocean crust is capable of supporting microbial communities, that iron oxidation and nitrate reduction may be important physiological characteristics of ocean crust microbes, and that heterogeneously distributed minerals in marine igneous rocks likely influence the distribution of microbial communities in the ocean crust.The subseafloor flow cell enrichment chambers were funded by a small grant from the Ocean Drilling Program. This work was also funded by NASA grant NNX08AO22G, NSF OCE 0727119 to C.G.W., NSF OCE 0452333 to S.M.S., and OCE‐0550713 and OCE‐0727952 to A.T.F., PSU, and OSU

A model for the coupling of α-helix and tertiary contact formation

Peptides corresponding to excised α-helical segments of natural proteins can spontaneously form helices in solution. However, peptide helices are usually substantially less stable in solution than in the structural context of a folded protein, because of the additional interactions possible between helices in a protein. Such interactions can be thought of as coupling helix formation and tertiary contact formation. The relative energetic contributions of the two processes to the total energy of the folded state of a protein is a matter of current debate. To investigate this balance, an extended helix-coil model (XHC) that incorporates both effects has been constructed. The model treats helix formation with the Lifson-Roig formalism, which describes helix initiation and propagation through cooperative local interactions. The model postulates an additional parameter representing participation of a site in a tertiary contact. In the model, greater helix stability can be achieved through combinations of these short-range and long-range interactions. For instance, stronger tertiary contacts can compensate for helices with little intrinsic stability. By varying the strength of the nonlocal interactions, the model can exhibit behavior consistent with a variety of qualitative models describing the relative importance of secondary and tertiary structure. Moreover, the model is explicit in that it can be used to fit experimental data to individual peptide sequences, providing a means to quantify the two contributions on a common energetic basis

Repeat protein architectures predicted by a continuum representation of fold space

It is an open question whether nature has utilized all possible protein folds. For a simple protein architecture, the helical repeats, we report a method to address this question based on a mapping between the set of repetitive curves and a space of parameters specifying the curve. The exploration of the parameter space for a particular architecture enables a systematic exploration of the fold space for that protein architecture. In a planar subspace of the parameter space of helical repeats we have identified points corresponding to both naturally occurring folds and potential folds not observed so far

The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress in Vivo Indicates That These Chaperones Protect a Wide Range of Cellular Functions

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function

The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system