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Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.

Accumulation of class I small heat shock proteins (sHSPs) is induced by the proline analog, azetidine-2-carboxylic acid (Aze) in soybean seedlings to a level similar to that induced by exposure to 40 degrees C. However, only the treatment with 10 mM Aze for 6 h and subsequently with 10 mM proline for 24 h protected the seedlings from damage during subsequent exposure to 45 degrees C as assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. A chaperone activity assay showed that the purified class I sHSPs induced by Aze were functional in vitro and protected proteins from thermal denaturation. Amino acid composition analysis indicated that Aze was not incorporated into de novo synthesized class I sHSPs. Accumulation of class I sHSPs in the soluble post-ribosomal supernatant fraction was found to be important for acquisition of thermotolerance. We suggest that both the accumulation of class I sHSPs and their presence in the soluble fraction are important for establishment of thermotolerance.

Equilibrium studies of guanidine hydrochloride (GdnHCl)-induced unfolding of dimeric arginine kinase (AK) from sea cucumber have been performed by monitoring by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphthalene-8sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking. The unfolding is a multiphasic process involving at least two dimeric intermediates. The first intermediate, I1, which exists at 0-0.4 M GdnHCl, is a compact inactive dimer lacking partial global structure, while the second dimeric intermediate, I2, formed at 0.5-2.0 M GdnHCl, possesses characteristics similar to the globular folding intermediates described in the literature. The whole unfolding process can be described as follows: (1) inactivation and the appearance of the dimeric intermediate I1; (2) sudden unwinding of I1 to another dimeric intermediate, I2; (3) dissociation of dimeric intermediate I2 to monomers U. The refolding processes initiated by rapid dilution in renaturation buffers indicate that denaturation at low GdnHCl concentrations (below 0.4 M GdnHCl) is reversible and that there seems to be an energy barrier between the two intermediates (0.4-0.5 M GdnHCl), which makes it difficult for AK denatured at high GdnHCl concentrations (above 0.5 M) to reconstitute and regain its catalytic activity completely.

The objective of the present study was to assess if cervical ripeness could be quantified by measuring the percentage of denaturation of the collagen network of the stromal layer. Biopsy specimens from the caudal part of the cervix were obtained from nine pluriparous cows between Days 149 and 157 of gestation (second-trimester biopsy), at exactly Day 275 of gestation (term biopsy), and shortly after calving (calving biopsy). The samples were divided into a superficial stromal part and a deep stromal part. The water content was derived from the weight of the samples before and after lyophilization. A colorimetric assay was used to assess the percentage of collagen denaturation by determining the extinction at 570 nm of hydroxyproline released from alpha-chymotrypsine-treated samples. By incorporating a hydroxyproline standard series in the measurements, the insoluble collagen content (mug/mg dry wt) as well as the insoluble collagen concentration (mug/mg wet wt) could be derived. The water content of both layers of the cervix significantly increased between midpregnancy and parturition (P < 0.01). The insoluble collagen content and the insoluble collagen concentration were significantly increased at term (P < 0.01 and P < 0.05, respectively) but were significantly decreased at calving (P < 0.05 and P < 0.01, respectively). Both parameters showed no significant differences between the superficial and deep stromal layer, and they were significantly correlated with each other. A significant increase in the percentage denaturation of the deep stromal layer occurred between the second trimester and term pregnancy (P < 0.01), whereas at calving, the percentage denaturation had not significantly increased compared to term. The percentage of collagen denaturation of the superficial stromal layer did not significantly change with stage of gestation or at parturition. Our findings indicate that cervical ripening is a combination of increased collagen synthesis and increased percentage of collagen denaturation, whereas at calving, an increased digestion of the denatured collagen leads to increased collagen loss from the cervical connective tissue. The finding that cervical ripening mainly takes place in the deep stromal layer of the cervix emphasizes the importance of a detailed description of the tissue sampling sites for a proper interpretation of the results obtained from biochemical studies of the cervix.

Neocarzinostatin (NCS) is a seven-stranded beta-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al. (2001) Protein Sci., 10, 2228-2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this beta-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the beta-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple 'large-to-small' substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.