The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Highly concentrated human recombinant interleukin-1 receptor antagonist (IL-1ra) aggregates at elevated temperature without perturbation in its secondary structure. The protein aggregation can be suppressed depending on the buffer ionic strength and the type of anion present in the sample solution. Phosphate is an approximately 4-fold weaker suppressant than either citrate or pyrophosphate on the basis of the measured protein aggregation rates. This is in agreement with the strength of protein-anion interactions at the IL-1ra single anion-binding site as judged by the estimated dissociation constant values of 2.9 mM, 3.8 mM, and 13.7 mM for pyrophosphate, citrate, and phosphate, respectively. The strength of binding also correlates with the anion size and with the number of ionized groups available per molecule at a given pH. Affinity probing of IL-1ra with methyl acetyl phosphate (MAP) in combination with proteolytic digestion and mass spectral analysis show that an anion-binding site location on the IL-1ra surface is contributed by lysine-93 and lysine-96 of the loop 84-98 as well as by lysine-6 of the unstructured N-terminal region 1-7. The replacement of lysine-93 with alanine by site-directed mutagenesis results in dramatically suppressed IL-1ra aggregation. Furthermore, when the unstructured N-terminal region of IL-1ra is removed by limited proteolysis, a 2-fold increase in the time course of the aggregation lag phase is observed for the truncated protein. An anion-controlled mechanism of IL-1ra aggregation is proposed by which the anion competition for the protein cationic site prevents formation of intermolecular cation-pi interactions and, thus, interferes with the protein asymmetric self-association pathway.

No abstract

Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using highresolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS-protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solventexposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation.

No abstract

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.The reversible conversion of many flavoenzymes to their corresponding apoproteins can be achieved under relatively mild conditions either by the perturbation of the protein structure in low pH solution, containing high concentration of salt, or by the addition of denaturating agents such as urea or guanidinium hydrochloride (1). This approach has been successfully employed to study flavoprotein active site environment and/or the mechanism of catalysis by using various flavin analogs as spectral or chemically reactive affinity probes (2-5). However, in some cases no viable apoprotein can be prepared from the corresponding flavoenzyme due to irreversible changes in the protein structure that result in the lack of either ligand binding and/or protein solubility. In addition, the yield and stability of the apoprotein depends largely on the method of its preparation. In fact, the treatment of porcine lipoamide dehydrogenase with 5 M guanidinium hydrochloride results in unstable and poorly reconstitutable apoprotein, while the combination of low pH-high salt concentration and hydrophobic interaction chromatography leads to a high yield of its fully reconstitutable form (3, 6, 7). Still, the strategy for the development of a suitable method of apoprotein preparation from a particular flavoenzyme remains largely empirical and is simply based on screening of the various known procedures. It is conceivable that the problem of obtaining reconstitutable apoprotein is directly related to other basic problems such as the control of protein stability in vitro by change in its microenvironment (denaturants, stabilizers, pH, etc.) (8-11) and the existence of intermediate states of protein during unfolding-refolding processes (12, 13).The subject of the present study was Crotalus adamanteus L-amino acid oxidase (LAAO). LAAO is a dimeric glycoprotein containing two molecules of noncovalently bound flavin adenine dinucleotide (FAD) per protein molecule and catalyzes oxidative deamination of various, but predominantly hydrophobic, L-amino acids (14). Our particular interest was raised by the fact that this enzyme has a highly sensitive active site, such that it undergoes reversible pH-or temperaturedependent inactiva...

The replacement of histidine 307 with leucine in pig kidney D-amino acid oxidase perturbs its active site conformation accompanied by dramatic losses in proteinflavin interactions and enzymatic activity. However, the negative effect of this mutation on the holoenzyme structure is essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flavin affinity. Further analysis revealed that glycerol assists in the rearrangement of the protein toward its holoenzyme-like conformation together with reduction in the solventaccessible protein hydrophobic area as demonstrated by limited proteolysis and use of affinity and hydrophobic probes. A substantial decrease in the protein-flavin interactions was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest that the perturbation of the D-amino acid oxidase active site is due to the nonpolar nature of the mutation whose negative impact on the holoenzyme structure can be overcome by glycerol-induced strengthening of protein internal hydrophobic interactions.The versatile properties of the flavin prosthetic group located in the active site of flavoenzymes have been successfully used over the years to study enzymatic redox mechanisms, including flavin-mediated activation of molecular oxygen (1, 2). In addition, the spectral redox properties and the reactivity of the flavin can be greatly manipulated by its modification with various chemical groups, a feature that has been particularly useful for structure functional analysis of flavoproteins (3-8). Flavoproteins also can be suitable models to study certain basic problems, such as protein stability, folding, and the effect of site-directed mutagenesis on protein conformation, since even the fine structural rearrangements within the protein active site can affect the flavin microenvironment and therefore be detected by perturbation of the binding mode and the spectral properties of the flavin. Recently, we employed this concept to demonstrate that glycerol, acting in vitro as a chemical chaperone, can assist in the proper refolding of the flavoprotein L-amino acid oxidase from its compact equilibrium intermediates and suggested the hydrophobic effect as a dominating force in this process (9). These findings prompted us to further investigate whether glycerol, in a similar manner, can induce proper adjustments of protein conformation perturbed by sitedirected mutagenesis, using a His-307 3 Leu (H307L) recombinant mutant of pig kidney D-amino acid oxidase (DAAO) 1 (10) as a model system. His-307 is one of two residues (the other is Tyr-228) undergoing affinity labeling with the D-propargylglycine-suicide substrate of pig kidney DAAO (11-13). Furthermore, the replacement of His-307 with Leu greatly reduces the protein affinity for FAD so that DAAO can be isolated only as an inactive apoprotein (14, 15). Although this is an indication that the presence of His-307 is important for protein...