Jeffrey W. Nelson

Secondary structure of the 33 kDa, extrinsic protein of photosystem II: a far-UV circular dichroism study

The 33 kDa extrinsic protein of Photosystem II is an important component of the oxygen-evolving apparatus which functions to stabilize the manganese cluster at physiological chloride concentrations and to lower the calcium requirement for oxygen evolution. Chou-Fasman analysis of the amino-acid sequence of this protein suggests that this component contains a high proportion of alpha-helical structure and only relatively small amounts of beta-sheet structure. A computational study using more sophisticated techniques (Beauregard, M. (1992) Environ. Exp. Bot. 32, 411-429) concluded that the protein contained little periodically ordered secondary structure. In this study, we have directly measured the relative proportions of secondary structure present in the 33 kDa protein using far-ultraviolet circular dichroism spectroscopy. Our results indicate that, in solution, this protein contains a large proportion of beta-sheet structure (38%) and relatively small amounts of alpha-helical structure (9%). A structural model of the 33 kDa protein based on a constrained Chou-Fasman analysis (Teeter, M.M. and Whitlow, M (1988) Proteins 4, 262-273) is presented.

Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTTSH SH), and oxidized dithiothreitol (DTTS S) has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25°C for the reaction GSSG + DTTSH SH ⇌ 2GSH + S S varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.

Strategies for selecting mutation sites for methionine enhancement in the bean seed storage protein phaseolin

The complete three-dimensional structure of the bean seed storage protein phaseolin was generated from α-carbon coordinates by using molecular mechanic calculations. This structure was used as a template to simulate modifications aimed at increasing the methionine content of phaseolin. A hydrophilic, methionine-rich looping insert sequence was designed. Simulated mutagenesis shows that the insert might be accommodated in turn and loop regions of the protein, but not within an α-helix. Methionine content was also increased by the replacement of hydrophobic amino acids with methionine in the central core β-barrels of the phaseolin protein. Calculations indicated that methionine can effectively replace conserved or variant leucine, isolecuine, and valine residues. However, alanine residues were much more sensitive to substitution, and demonstrated high variability in the effects of methionine replacement. Introduction of multiple substitutions in the barrel interior demonstrated that the replaced residues could interact favorably to relieve local perturbations caused by individual substitutions. Molecular dynamics simulations were also utilized to study the structural organization of phaseolin. The calculations indicate that there are extensive packing interactions between the major domains of phaseolin, which have important implications for protein folding and stability. Since the proposed mutant proteins can be produced and studied, the results presented here provide an ideal test to determine if there is a correlation between the effects obtained by computer simulation and the effects of the mutations on the protein structure expressedin vivo.

A complete nucleolin cDNA sequence from Xenopus laevis

Nucleolin is a nucleolar specific protein that is thought to play a role in the processing of pre-ribosomal RNA as ribosomes are assembled (1). A full length Xenopus nucleolin cDNA was constructed from overlapping sequences recovered from an ovary cDNA library. The intact cDNA is 2443 bp in length with the ATG start codon at ops 64-66 and the TGA stop codon at bps 2017-2019. A stop codon at bps 19-21 is in frame with the ATG start codon suggesting that the cDNA is complete. The deduced protein is 651 amino acid residues in length (Fig. 1). Like other vertebrate forms of nucleolin, the amino terminal third of Xenopus nucleolin contains alternating basic and acidic domains, while the carboxy two-thirds consists of four RNAbinding domains and a glycine/arginine rich domain (2). The largest basic domain within the amino terminal third of chicken (3), CHO (4), and Xenopus nucleolin (underlined in Fig. 1) are compared in Fig. 2. Both nucleolin TPA/GKK motifs (underlined in Fig. 2) and histone HI SPKK motifs are substrates for p34°* kinase (5,6), and we have previously shown with a monoclonal antibody, that various vertebrate nucleolin proteins share an antigenic epitope with histone HI (7). The largest basic domain from the amino terminal third of Xenopus nucleolin compares well with sea urchin gonadal histone HI (Fig. 3A), specifically, the spacing of proline residues (asterisks). Similar periodicities of proline residues are evident when the largest basic domain within the amino terminal region of CHO nucleolin is compared to Xenopus histone HI A (Fig. 3B). The similar periodicity of proline residues in nucleolin and histone HI suggests similar structures, perhaps reverse /3-turns (8, 9) or kinking alpha helices (10, 11).

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.

Extensive modifications for methionine enhancement in the β-barrels do not alter the structural stability of the bean seed storage protein phaseolin

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. AnEscherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded inE. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61°C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in theβ-barrel and loop structures, with extremely small effects on protein stability.