Chaperone function of two small heat shock proteins from maize.

Small heat shock proteins (sHsps) are molecular chaperones that protect cells from the effect of heat and other stresses. Some sHsps are also expressed at specific stages of development. In plants different classes of sHsps are expressed in the various cellular compartments. While the Class I (cytosolic) sHsps in wheat and pea have been studied extensively, there are fewer experimental data on Class II (cytosolic) sHsps, especially in maize. Here we report the expression and purification of two Class II sHsps from Zea mays ssp. mays L. (cv. Oh43). The two proteins have almost identical sequences, with the significant exception of an additional nine-amino-acid intervening sequence near the beginning of the N-terminus in one of them. Both ZmHsp17.0-CII and ZmHsp17.8-CII oligomerize to form dodecamers at temperatures below heat shock, and we were able to visualize these dodecamers with TEM. There are significant differences between the two sHsps during heat shock at 43°C: ZmHsp17.8-CII dissociates into smaller oligomers than ZmHsp17.0-CII, and ZmHsp17.8-CII is a more efficient chaperone with target protein citrate synthase. Together with the previous observation that ZmHsp17.0-CII but not ZmHsp17.8-CII is expressed during development, we propose different roles in the cell for these two sHsps.

An unusual dimeric small heat shock protein provides insight into the mechanism of this class of chaperones.

Small heat shock proteins (sHSPs) are virtually ubiquitous stress proteins that are also found in many normal tissues and accumulate in diseases of protein folding. They generally act as ATP-independent chaperones to bind and stabilize denaturing proteins that can be later reactivated by ATP-dependent Hsp70/DnaK, but the mechanism of substrate capture by sHSPs remains poorly understood. A majority of sHSPs form large oligomers, a property that has been linked to their effective chaperone action. We describe AtHsp18.5 from Arabidopsis thaliana, demonstrating that it is dimeric and exhibits robust chaperone activity, which adds support to the model that suboligomeric sHSP forms are a substrate binding species. Notably, like oligomeric sHSPs, when bound to substrate, AtHsp18.5 assembles into large complexes, indicating that reformation of sHSP oligomeric contacts is not required for assembly of sHSP-substrate complexes. Monomers of AtHsp18.5 freely exchange between dimers but fail to coassemble in vitro with dodecameric plant cytosolic sHSPs, suggesting that AtHsp18.5 does not interact by coassembly with these other sHSPs in vivo. Data from controlled proteolysis and hydrogen-deuterium exchange coupled with mass spectrometry show that the N- and C-termini of AtHsp18.5 are highly accessible and lack stable secondary structure, most likely a requirement for substrate interaction. Chaperone activity of a series of AtHsp18.5 truncation mutants confirms that the N-terminal arm is required for substrate protection and that different substrates interact differently with the N-terminal arm. In total, these data imply that the core α-crystallin domain of the sHSPs is a platform for flexible arms that capture substrates to maintain their solubility.

Conformation and hydrogen bonding for the bicyclic compound 3-thiabicyclo[3.2.0]heptane-6,7-dicarboxylic acid 3,3-dioxide.

The initial goal of this work was to verify the geometry of the product of a photochemical reaction, viz. the title compound, C(8)H(10)O(6)S, (II). Our crystallographic study firmly establishes the cis-anti-cis nature of the substituents on the cyclobutane ring. The geometry is also designated as exo, where exo signifies that the five-membered ring is on the opposite side of the central cyclobutane ring from the carboxylic acid substituents. The structure determination reveals two molecules, A and B, in the asymmetric unit that display substantially different conformations of the bicyclic core: the cyclobutane ring puckering angles are 22 and 3 degrees , and the sulfolane ring conformations are twist (S-exo) and envelope (S-endo). Intrigued by this variation, we then compared the conformations of other molecules in the Cambridge Structural Database that have sulfolane rings fused to cyclobutane rings. In this class of compound, there are five examples of saturated cyclobutane rings, with ring puckering angles ranging from 3 to 35 degrees . The sulfolane rings were more similar: four of the six molecules exhibit envelope conformations with S-endo, as in molecule B of (II). Despite the conformational differences, the hydrogen-bonding scheme for both molecules is similar: carboxyl -OH groups form hydrogen bonds with carboxyl and sulfone O atoms. Alternating A and B molecules joined by hydrogen bonds between sulfone O atoms and carboxyl -OH groups form parallel chains that extend in the ac plane. Other hydrogen bonds between the carboxyl groups link the chains along the b axis.

Ring opening of pyridines: the pseudo-cis and pseudo-trans isomers of tetra-n-butylammonium 4-nitro-5-oxo-2-pentenenitrilate.

The reaction of 2-chloro-5-nitropyridine with two equivalents of base produces the title carbanion as an intermediate in a ring-opening/ring-closing reaction. The crystal structures of the tetra-n-butylammonium salts of the intermediates, C(16)H(36)N(+).C(5)H(3)N(2)O(3)(-), revealed that pseudo-cis and pseudo-trans isomers are possible. One crystal structure displayed a mixture of the two isomers with approximately 90% pseudo-cis geometry and confirms the structure predicted by the S(N)(ANRORC) mechanism. The pseudo-cis intermediate undergoes a slow isomerization over a period of months to the pseudo-trans isomer, which does not have the appropriate geometry for the subsequent ring-closing reaction. The structure of the pure pseudo-trans isomer is also reported. In both isomers, the negative charge is highly delocalized, but relatively small differences in C-C bond distances indicate a system of conjugated double bonds with the nitro group bearing the negative charge. The packing of the two unit cells is very similar and largely determined by the interactions between the planar carbanion and the bulky tetrahedral cation.

Structures of the intermediates formed in the ring-opening reaction of 2-chloro-3-nitropyridine.

The reaction of 2-chloro-3-nitropyridine with two equivalents of hydroxide ion was studied by NMR and X-ray crystallography. On the basis of NMR coupling constants, the originally formed ring-opened intermediate is the pseudo-cis form, as predicted by the SN(ANRORC) mechanism. However, the first intermediate is unstable and isomerizes to a second intermediate, which was isolated. The pseudo-trans geometry of the second intermediate [3-pentenenitrile, 2-nitro-5-oxo, ion(-1), sodium] explains why additional base does not lead to the ring-closing reaction as observed with 2-chloro-5-nitropyridine.

2,5-dichloro-2,5-dihydrothiophene 1-oxide: determination of the complete stereochemistry.

The title five-membered heterocycle, C4H4Cl2OS, adopts an envelope conformation with the S atom at the tip of the flap. All three ring substituents, viz. the sulfoxide O atom and the two Cl atoms, are cis to each other. The two C atoms alpha to the sulfoxide group are also bonded to chlorine. The electron-withdrawing chlorine substituents give rise to weak C-H...O hydrogen bonds with the sulfoxide O atom of a symmetry-related molecule [H...O = 2.44 (2) and 2.61 (2) A, C...O = 3.143 (3) and 3.302 (2) A and C-H...O = 129.9 (19) and 135.1 (19) degrees ]. There is also a possible weak C-H...Cl interaction. Chains of molecules held together by these weak interactions run parallel to the a axis.

The nature of interactions in the ionic crystal of 3-pentenenitrile, 2-nitro-5-oxo, ion(-1), sodium.

The hybrid variation -- perturbation many-body interaction energy decomposition scheme has been applied to analyze the physical nature of interactions in the ionic 3-pentenenitrile, 2-nitro-5-oxo, ion(-1), sodium crystal, which can be regarded as a model for a large group of aromatic quaternary nitrogen salts. In the crystal structure the sodium ions and water molecules of adjacent unit cells form a positively charged "inorganic layer" with the sodium ions clustered together along the ab faces with the organic (negative) part in between. This puzzling crystal packing is due to a strong favorable interaction between the water molecule and the sodium ions and a substantial charge transfer from the carbanions that balances out the destabilizing sodium-sodium ion repulsion. Although the majority of cohesion energy of the crystal structure comes from the electrostatic interactions of ions, the resulting net stabilization also depends heavily on the nonadditive delocalization components, due to a counterbalance between the two-body delocalization and exchange effects. The estimated nonadditivity of interactions varies between 12% and 22%.