Directed evolution of the suicide protein O6-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability.

Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially of the alkylated protein, suggesting that the reactivity of the suicide enzyme can be influenced by stabilizing the product of the irreversible reaction. Whereas wild-type hAGT is rapidly degraded in cells after alkyl transfer, the high stability of benzylated SNAP-tag prevents proteolytic degradation. Our data indicate that the intrinstic stability of a key α helix is an important factor in triggering the unfolding and degradation of wild-type hAGT upon alkyl transfer, providing new insights into the structure-function relationship of the DNA repair protein.

Fura-2FF-based calcium indicator for protein labeling.

We describe the synthesis and fluorescence properties of a Fura-2FF-based fluorescent Ca(2+) indicator that can be covalently linked to SNAP-tag fusion proteins and retains its Ca(2+) sensing ability after coupling to protein.

Indo-1 derivatives for local calcium sensing.

The role of calcium in signal transduction relies on the precise spatial and temporal control of its concentration. The existing means to detect fluctuations in Ca2+ concentrations with adequate temporal and spatial resolution are limited. We introduce here a method to measure Ca2+ concentrations in defined locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1 to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in single cells dissociated from muscle of adult mice expressing a nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by shifted excitation and emission ratioing of confocal microscopic images of fluorescence. After permeabilizing the plasma membrane, changes in the bathing solution induced corresponding changes in nuclear [Ca2+] that were readily detected and used for a preliminary calibration of the technique. This work thus demonstrates the synthesis and application of SNAP-tag-based Ca2+ indicators that combine the spatial specificity of genetically encoded calcium indicators with the advantageous spectroscopic properties of synthetic indicators.

Reaction geometry and thermostable variant of pyranose 2-oxidase from the white-rot fungus Peniophora sp.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen; glucose, for example, is oxidized at carbon 2. The structure of pyranose 2-oxidase with the reaction product 2-keto-beta-d-glucose bound in the active center is reported in a new crystal form at 1.41 A resolution. The binding structure suggests that the alpha-anomer cannot be processed. The binding mode of the oxidized product was used to model other sugars accepted by the enzyme and to explain its specificity and catalytic rates. The reported structure at pH 6.0 shows a drastic conformational change in the loop of residues 454-461 (loop 454-461) at the active center compared to that of a closely homologous enzyme analyzed at pH 4.5 with a bound acetate inhibitor. In our structures, the loop is highly mobile and shifts to make way for the sugar to pass into the active center. Presumably, loop 454-461 functions as a gatekeeper. Apart from the wild-type enzyme, a thermostable variant was analyzed at 1.84 A resolution. In this variant, Glu542 is exchanged for a lysine. The observed stabilization could be a result of the mutated residue changing an ionic contact at a comparatively weak interface of the tetramer.

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore.

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.

Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.

The expression of outer membrane proteins for crystallization.

The production of sufficient amounts of chemically and conformationally homogenous protein is a major requirement for successful crystallization and structure determination. With membrane proteins, this constitutes a particular problem because the membrane volume is limited and the organisms are usually very sensitive to changes in membrane properties brought about by massive protein insertion. Moreover, the extraction of membrane proteins from the membrane with detergents is generally a harsh treatment, which gives rise to conformational aberrations. A number of successful procedures for functional expression followed by purification are reviewed here together with nonfunctional expression into inclusion bodies and subsequent (re)folding to produce functional proteins. Most of the data are for prokaryotic outer membrane proteins, but the outer membrane proteins of eukaryotic organelles are also considered as they do show similar features.

Asymmetric conductivity of engineered porins.

Positively charged peptide segments of 16 and 18 residues were inserted at a periplasmic turn of the porin from Rhodobacter blasticus in order to form an electric field-dependent plug. The X-ray diffraction analysis of a mutant confirmed that the structure of the porin had remained intact and that the insert was mobile. Incorporation experiments of single molecules into lipid bilayers showed that the distribution of electric conduction increments depended on the field polarity. The observed distributions are explained if the porin molecules enter the bilayer preferentially with their periplasmic surface first. Furthermore, the conduction of membrane-incorporated porin mutants changed reproducibly on field reversal showing asymmetries of reverse similar 15%, while the wild-type remained constant. This asymmetry is most likely caused by the electric field pressing the charged insert onto the pore eyelet in one field direction and removing it from the eyelet in the other. The results encourage attempts to improve the inserts in order to eventually reach diode characteristics.