Clarence E. Schutt

The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes.

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.

The crystal structure of a major allergen from plants.

BACKGROUND Profilins are small eukaryotic proteins involved in modulating the assembly of actin microfilaments in the cytoplasm. They are able to bind both phosphatidylinositol-4,5-bisphosphate and poly-L-proline (PLP) and thus play a critical role in signaling pathways. Plant profilins are of interest because immunological cross-reactivity between pollen and human profilin may be the cause of hay fever and broad allergies to pollens. RESULTS The determination of the Arabidopsis thaliana profilin isoform I structure, using multiwavelength anomalous diffraction (MAD) to obtain structure-factor phases, is reported here. The structure of Arabidopsis profilin is similar to that of previously determined profilin structures. Conserved amino acid residues in profilins from plants, mammals, and lower eukaryotes are critically important in dictating the geometry of the PLP-binding site and the overall polypeptide fold. The main feature distinguishing plant profilins from other profilins is a solvent-filled pocket located in the most variable region of the fold. CONCLUSIONS Comparison of the structures of SH3 domains with those of profilins from three distinct sources suggests that the mode of PLP binding may be similar. A comparison of three profilin structures from different families reveals only partial conservation of the actin-binding surface. The proximity of the semi-conserved actin-binding site and the binding pocket characteristic of plant profilins suggests that epitopes encompassing both features are responsible for the cross-reactivity of antibodies between human and plant profilins thought to be responsible for type I allergies.

The enhancement of PCR amplification by low molecular-weight sulfones.

DNA amplification by polymerase chain reaction (PCR) is frequently complicated by the problems of low yield and specificity, especially when the GC content of the target sequence is high. A common approach to the optimization of such reactions is the addition of small quantities of certain organic chemicals, such as dimethylsulfoxide (DMSO), betaine, polyethylene glycol and formamide, to the reaction mixture. Even in the presence of such additives, however, the amplification of GC-rich templates is often ineffective. In this paper, we introduce a novel class of PCR-enhancing compounds, the low molecular-weight sulfones, that are effective in the optimization of high GC template amplification. We describe here the results of an extensive structure-activity investigation in which we studied the effects of a series of six different sulfones on PCR amplification. We identify two sulfones, sulfolane and methyl sulfone, that are especially potent enhancers of high GC template amplification, and show that these compounds often outperform DMSO and betaine, two of the most effective PCR enhancers currently used. We conclude with a brief discussion of the role that the sulfone functional group may play in such enhancement.

A Comparative Structural Analysis of the ADF/Cofilin Family

Actin‐depolymerizing factor (ADF) and cofilin define a family of actin‐binding proteins essential for the rapid turnover of filamentous actin in vivo. Here we present the 2.0 Å crystal structure of Arabidopsis thaliana ADF1 (AtADF1), the first plant crystal structure from the ADF/cofilin (AC) family. Superposition of the four AC isoform structures permits an accurate sequence alignment that differs from previously reported data for the location of vertebrate‐specific inserts and reveals a contiguous, vertebrate‐specific surface opposite the putative actin‐binding surface. Extending the structure‐based sequence alignment to include 30 additional isoforms indicates three major groups: vertebrates, plants, and “other eukaryotes.” Within these groups, several structurally conserved residues that are not conserved throughout the entire AC family have been identified. Residues that are highly conserved among all isoforms tend to cluster around the tryptophan at position 90 and a structurally conserved kink in α‐helix 3. Analysis of surface character shows the presence of a hydrophobic patch and a highly conserved acidic cluster, both of which include several residues previously implicated in actin binding. Proteins 2000;41:374–384.

Tropomyosin and Gelsolin Cooperate in Controlling the Microfilament System

Tropomyosin has been shown to cause annealing of gelsolin-capped actin filaments. Here we show that tropomyosin is highly efficient in transforming even the smallest gelsolin-actin complexes into long actin filaments. At low concentrations of tropomyosin, the effect of tropomyosin depends on the length of the actin oligomer, and the cooperative nature of the process is a direct indication that tropomyosin induces a conformational change in the gelsolin-actin complexes, altering the structure at the actin (+) end such that capping by gelsolin is abolished. At increased concentrations of tropomyosin, heterodimers, trimers, and tetramers are converted to actin filaments. In addition, evidence is presented demonstrating that gelsolin, once removed from the (+) end of the actin, can reassociate with the newly formed tropomyosin-decorated actin filaments. Interestingly, the binding of gelsolin to the tropomyosin-actin filament complexes saturates at 2 gelsolin molecules per 14 actin and 2 tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along the filament. These observations support the view that both tropomyosin and gelsolin are likely to have important functions in addition to those proposed earlier.

The oscillation method for crystals with very large unit cells

The processing of data collected by oscillation photography from crystals with very large unit cells (average cell constant ∼ 250 A) requires important modifications of standard methods. In particular, it is often necessary to correct partially recorded intensities to their fully recorded equivalents. A procedure is described for accurate determination of relevant parameters (crystal setting, cell constants). It relies on the redundancy present in most data-collection strategies, which yields fully recorded, symmetry-related counterparts of a number of partially recorded reflections. A detailed description of one example (tomato bushy stunt virus) is presented, with complete intensity statistics.

Structural changes in profilin accompany its binding to phosphatidylinositol 4,5-bisphosphate

The effect on the structure of profilin of phosphatidylinositol 4,5‐bisphosphate (PIP2) binding was probed by fluorescence and circular dichroism (CD) spectroscopy, Fluorescence of Trp3 and Trp31 of profilin at 292 nm showed a linear decrease in solution emission at 340 nm as PIP2/profilin was increased from 0 to 80:1, apparently due to a static quenching mechanism involving formation of a nonfluorescent PIP2/profilin complex. CD spectra revealed an increase of up to 3.3‐fold in the molar ellipticity at 222 nm for profilin as it binds PIP2, as well as changes in the Cotton effect between 250 and 310 nm. These results are consistent with a possible increase in the α‐helix content or profilin triggered by the binding or PIP2.

Affinity chromatography-based purification of profilin:actin.

Publisher Summary In nonmuscle cells, as much as 50% of the cytoplasmic actin appears to exist in the nonfilamentous state, a significant portion of which is associated with profilin in a 1:1 complex called profilin:actin or profilactin. Originally, profilactin from mammalian nonmuscle tissue was purified using ammonium sulfate fractionation followed by successive chromatographic separations on Cγ alumina gel, DEAE-Sephadex, and Sephadex G-100. Additional chromatography on hydroxylapatite could then be employed to separate the profilin:β-actin and profilin:γ-actin isoforms. Recently, the affinity of profilin:actin to a poly(L-proline)-Sepharose matrix has been used to develop an alternative single-step procedure for purifying profilin:actin from nonmuscle tissue extracts. This chapter describes the purification of profilin:actin from mammalian nonmuscle tissue by poly(L-proline)-Sepharose and gel-filtration chromatography, and the separation of profilin:β-actin from profilin:γ-actin using hydroxylapatite chromatography. Methods for the separation of profilin from actin and the crystallization of profilin:actin is also discussed.

Structural aspects of actin-binding proteins

The three-dimensional structures of myosin subfragment 1 (S1), gelsolin segment 1 complexed with alpha-actin, villin fragment 14T, Acanthamoeba profilin-I, and bovine profilin complexed with beta-actin were completed last year. Together, they expand our understanding of the structural organization of actin-binding proteins. In addition, the segment 1 and bovine profilin complexes provide atomic-level descriptions of their interfaces with actin.

Mutations in β-actin: influence on polymer formation and on interactions with myosin and profilin

Two β‐actin mutants, one with proline 38 replaced with alanine (P38A) and the other with cysteine‐374 replaced with serine (C374S), as well as the wild‐type β‐actin, were expressed in the yeast, S. cerevisiae, purified to homogeneity, and analyzed in vitro for polymerizability and interaction with DNase I, myosin, and profilin. Both mutations interfered with the polymerization of the actin, and with its interaction with myosin. The C374S mutation had the most pronounced effect; it reduced the polymerizability of the actin, abolished its binding to profilin, and filaments containing this mutation moved at reduced rates in the in vitro ‘motility assay’. The ATPase activity measured in solutions containing myosin subfragment 1 was similar for both the mutant and wild‐type actins.