Christos Tziatzios

Projection structure and oligomeric properties of a bacterial core protein translocase

The major route for protein export or membrane integration in bacteria occurs via the Sec‐dependent transport apparatus. The core complex in the inner membrane, consisting of SecYEG, forms a protein‐conducting channel, while the ATPase SecA drives translocation of substrate across the membrane. The SecYEG complex from Escherichia coli was overexpressed, purified and crystallized in two dimensions. A 9 Å projection structure was calculated using electron cryo‐microscopy. The structure exhibits P121 symmetry, having two asymmetric units inverted with respect to one another in the unit cell. The map shows elements of secondary structure that appear to be transmembrane helices. The crystallized form of SecYEG is too small to comprise the translocation channel and does not contain a large pore seen in other studies. In detergent solution, the SecYEG complex displays an equilibrium between monomeric and tetrameric forms. Our results therefore indicate that, unlike other known channels, the SecYEG complex can exist as both an assembled channel and an unassembled smaller unit, suggesting that transitions between the two states occur during a functional cycle.

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP′ part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of “natively unfolded” proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

Crystal structures and enzymatic properties of three formyltransferases from archaea: Environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N5‐formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate‐reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98°C) has recently been solved at 1.65 Å resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37°C) and from Archaeoglobus fulgidus (growth temperature optimum 83°C) at 1.9 Å and 2.0 Å resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was −32 for the M. kandleri enzyme, only −8 for the M. barkeri enzyme, and −11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3‐diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3‐diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting‐out salts.

Characterizing the Solution Properties of Supramolecular Systems by Analytical Ultracentrifugation

A modern version of a classical method of macromolecular and biological chemistry, analytical ultracentrifugation, can be used to study the state of association of supramolecular compounds in solution. In order to familiarize the reader with this technique, the apparatus for which is shown here, we describe application of three different types of ultracentrifuge experiment to a cobalt coordination array dissolved in acetone.

Studies on the partial specific volume of a poly(ethylene glycol) derivative in different solvent systems

The specific volume of charged supramolecular compounds dissolved in organic solvents varies considerably with the solvent system applied; in addition, it is influenced by the presence of salt. In this study we determined the specific volume of an uncharged molecule from the same molar mass range in order to find out whether it shows the same dependencies. To allow application of solvents of widely differing polarity, including water, a poly(ethylene glycol) derivative of molar mass 3,650 g/mol was used as a model system. The primary method applied for determining the specific volume was the buoyant density method, in which sedimentation equilibrium experiments using solvent mixtures of different density are performed and the specific volume is obtained as the reciprocal of that solvent density for which the compound is neutrally buoyant. A second method applied for determination of the specific volume was digital densimetry. We found that the strong influence of the solvent on the specific volume observed with charged compounds is also shown by the uncharged poly(ethylene glycol) derivative, the differences in the specific volume between different solvent systems amounting up to 15%; however, no significant dependence on the presence of salt was observed. We also found that, with the compound studied, a simple rule relating the specific volume and solvent polarity apparently does not exist.

Solution properties of supramolecular cobalt coordination arrays

A number of chemically related gridlike Co coordination arrays were studied by UV/vis absorption spectroscopy, electron microscopy and, in particular, analytical ultracentrifugation and partial specific volume measurements, in order to determine their solubility, stability and association behavior in a variety of organic solvents. As judged by the naked eye, solubilization of the compounds occurred instantaneously or at least within minutes. In contrast, the UV/ vis absorbance of the samples distinctly changed for hours or even days, depending on the compound in question. In some cases, the spectral changes indicated dissociation events, probably involving dissolution of clusters or microcrystals. This was supported by 1H NMR (on related Cd and Zn compounds) and electron microscopic observations at different time intervals after addition of the solvent. Under certain conditions, addition of 20–50 mM salt (necessary to obtain ultracentrifuge data not influenced by nonideal sedimentation behavior) again led to aggregation of the material. However, according to equilibrium sedimentation experiments in most solvents the solubilized Co coordination arrays finally were in the form of monomers, whereas in some solvents intermediate aggregates were predominant. Prolonged storage of the solubilized compounds at room temperature in most cases led to their decomposition or conversion. Reliably determining the partial specific volume, ν, of the compounds turned out to be the most difficult problem in our studies. Density measurements using a Paar density meter apparently suffered from disturbances (probably due to aggregation) at the relatively high compound concentrations required. v determinations applying the Edelstein—Schachman method to data collected in solvents of different density suffered from dependencies of v on the solvent. Combining measurements in nondeuterated and deuterated solvents (as in the original Edelstein—Schachman method) suffered from relatively low accuracy; in addition, it is applicable to a few solvents only. At present, weighted averages of the ν values from the different ultracentrifuge methods seem to yield the most reliable figures.

Metal ion assisted folding and supramolecular organization of a De Novo designed metalloprotein

This paper describes the supramolecular organization of a novel de novo designed metalloprotein, which consists of two N-terminal terpyridine modified coiled-coil protein folding motif sequences held together by an iron(II) ion. The self-assembly of the metalloprotein is the result of the interplay of metal ion complexation and protein folding, and can be manipulated by changes in concentration, temperature, and solvent. At low concentrations, folding and organization of the metalloprotein resembles that of the native coiled-coil peptide. Besides unimeric species, also dimeric and tetrameric metalloprotein assemblies were found. Several indications suggest that at least part of these unimeric species may exist as intramolecularly folded coiled-coils, however, unambiguous proof is lacking at the moment. At higher concentrations, folding and organization is dominated by the large octahedral [FeII(terpy)2] complexes (terpy = 2,2′:6′,2″-terpyridine) and considerable amounts of large, ill-defined aggregates are formed.

First GPC results of terpyridine based chain extended supramolecular polymers: comparison with viscosity and analytical ultracentrifugation

Abstract The molecular weight of an extended metallo-supramolecular polymer, based on a α,ω-bis-terpyridine-poly(ethylene glycol) polymer linked via ruthenium(II) ions, was determined by gel permeation chromatography, viscosimetry as well as analytical ultracentrifugation. An optimized GPC method was applied for the first time to this kind of chain extended supramolecular polymers and the obtained results showed a good agreement with viscosimetry and analytical ultracentrifugation. A chain extended polymer with an average molecular weight of around 140 000 was found. These results clearly demonstrate that well-soluble high-molecular-weight metal-containing coordination polymers that are based on well-defined telechelics can be synthesized and fully characterized.

The Molar Mass of an Active Photosystem I Complex from the Cyanobacterium Synechococcus PCC 7002

The molar mass of photosystem I (PS I) from the cyanobacterium Synechococcus PCC 7002 was determ ined by sedim entation equilibrium analysis in the analytical ultracentrifuge. For that purpose, the “trimeric” form of PS I was isolated and studied in three different nonionic detergents. After determining the partial specific volume of the protein/pigment complex, from the ultracentrifuge data, as (0.788 ± 0.010) ml g-1, Mr was obtained as 830,000 ± 60,000