Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes

Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature for the organism of origin. Increasing the ratio of sHSP to substrate during substrate denaturation decreased sHSP-substrate complex size, and accordingly, addition of substrate to pre-formed sHSP-substrate complexes increased complex size. However, the size of pre-formed sHSP-substrate complexes could not be reduced by addition of more sHSP, and substrate could not be observed to transfer to added sHSP, although added sHSP subunits continued to exchange with subunits in sHSPsubstrate complexes. Thus, although some number of sHSP subunits within complexes remain dynamic and may be important for complex structure/solubility, association of substrate with the sHSP does not appear to be similarly dynamic. These observations are consistent with a model in which ATP-dependent chaperones associate directly with sHSP-bound substrate to initiate refolding

The Weakest Bond: Experimental Observation of Helium Dimer

Helium dimer ion was observed after electron impact ionization of a supersonic expansion of helium with translational temperature near 1 mK. The dependence of the ion signal on source pressure, distance from the source, and electron kinetic energy was measured. The signal was determined to arise from ionization of neutral helium dimer

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

Influence of Retardation on the Vibrational Wave Function and Binding Energy of the Helium Dimer

Because of the extremely small binding energy of the helium dimer, the nuclear wave function is delocalized over an extremely large range of separations. One might therefore expect the properties of this extraordinary species to be sensitive to the potential at very large internuclear distances, r, where relativistic corrections to the usual van der Waals interaction may be important. We have estimated the effect of retardation, which changes the r-6 dependence of the potential to r-7 in the limit of large r, and have found that the binding energy and expectation value (r) are indeed significantly affected by its inclusion

Scaling, Multiscaling, and Nontrivial Exponents in Inelastic Collision Processes

We investigate velocity statistics of homogeneous inelastic gases using the Boltzmann equation. Employing an approximate uniform collision rate, we obtain analytic results valid in arbitrary dimension. In the freely evolving case, the velocity distribution is characterized by an algebraic large velocity tail, P(v,t) ~ v^{-sigma}. The exponent sigma(d,epsilon), a nontrivial root of an integral equation, varies continuously with the spatial dimension, d, and the dissipation coefficient, epsilon. Although the velocity distribution follows a scaling form, its moments exhibit multiscaling asymptotic behavior. Furthermore, the velocity autocorrelation function decays algebraically with time, A(t)= ~ t^{-alpha}, with a non-universal dissipation-dependent exponent alpha=1/epsilon. In the forced case, the steady state Fourier transform is obtained via a cumulant expansion. Even in this case, velocity correlations develop and the velocity distribution is non-Maxwellian.Comment: 10 pages, 3 figure