Polyoxometalate-stabilized, water dispersible Fe2Pt magnetic nanoparticles.

Magnetic Fe2Pt core-shell nanoparticles with 2 nm cores were synthesized with a monolayer coating of silicotungstate Keggin clusters. The core-shell composition is substantiated by structural analysis performed using high-resolution scanning transmission electron microscopy (HR-STEM) and small angle X-ray scattering (SAXS) in a liquid suspension. The molecular metal oxide cluster shell introduces an enhanced dispersibility of the magnetic Fe-Pt core-shell nanoparticles in aqueous media and thereby opens up new routes to nanoparticle bio-functionalization, for example, using pre-functionalized polyoxometalates.

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes.

Traditionally, the biological fluorination of complex biological systems like proteins is achieved through substitution of canonical amino acids or addition of fluorinated amino acids in the context of the standard genetic code. Ribosomal translation of monofluorinated amino acids into proteins often yields structures with minimal local changes in the interior but, on the same time, results in large global effects on characteristic features of the biopolymers (such as dramatically changed activity profile or folding stability). This is due to the novel and unique local interactions delivered by fluorine atoms such as (i) increase in the covalent radii (ii) changed polarities; (iii) changed hydrogen bond acceptor ability; (iv) altered water solubility as well as water ↔ organic solvent energy transfer. On the other hand, the biological incorporation of tri- or global fluorinated amino acids (such as trifluoroleucine, triflurovaline, and their hexafluoro counterparts, fluoromethionine and trifluoronorleucine etc.) represents still a challenge, as the natural structural scaffolds are optimized for hydrocarbon during evolution but not for fluorocarbon cores. Future work will be focused on the re-design of existing or de novo design of novel protein scaffolds capable of accommodating such building blocks into functional biologically active proteins and proteomes in the context of the viable cells.

Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy.

The photostability of the widely used autofluorescent proteins EGFP and EYFP and their fluorinated counterparts were compared by means of fluorescence correlation spectroscopy. We analyzed the reduction of the apparent diffusional time in analogy to fluorescence quenching in which the 'photon concentration' is treated as the quencher concentration. The quantum yields of photobleaching Phi(bl) of EYFP (6.1x10(-)(5)) and EGFP (8.2x10(-)(5)) are in agreement with the previously published values. Among the investigated mutants, EYFP has the highest photostability and there is an enhanced photobleaching in (2-F) Tyr-EYFP. It turns out that the chromophore fluorination has no significant influence on the photostability.

Incorporation of beta-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography.

beta-Selenolo[3,2-b]pyrrolyl-L-alanine that mimics tryptophan with the benzene ring of the indole moiety replaced by selenophene, was incorporated into human annexin V and barstar. This was achieved by fermentation and expression in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. The seleno- proteins were obtained in yields comparable to those of the wild-type proteins and exhibit full crystallographic isomorphism to the parent proteins, but expectedly show altered absorbance profiles and quenched tryptophan fluorescence. Since the occurrence of tryptophan residues in proteins is rare, incorporation of the electron-rich selenium-containing tryptophan surrogate into proteins represents a useful supplementation and even a promising novel alternative to selenomethionine for solving the phase problem in protein X-ray crystallography.

Proteins with beta-(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids.

L-beta-(Thieno[3,2-b]pyrrolyl)alanine and L-beta-(thieno[2,3-b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into recombinant proteins in response to the Trp UGG codons by fermentation in a Trp-auxotrophic Escherichia coli host strain using the selective pressure incorporation method. Related protein mutants expectedly retain the secondary structure of the native proteins but show significantly changed optical and thermodynamic properties. In this way, new spectral windows, fluorescence, polarity, thermodynamics, or pharmacological properties are inserted into proteins. Such an engineering approach by translational integration of synthetic amino acids with a priori defined properties, as shown in this study, proved to be a novel and useful tool for protein rational design.

Noninvasive tracing of recombinant proteins with "fluorophenylalanine-fingers".

High-level residue-specific replacement of phenylalanine residues in recombinant human annexin V and azurin from Pseudomonas aeruginosa with o-fluorophenylalanine, m-fluorophenylalanine, and p-fluorophenylalanine has been achieved using the selective pressure incorporation method. Incorporation was confirmed analytically and by UV spectroscopy while the secondary and tertiary structures of these protein mutants in solution remained unchanged upon the effected substitutions. Fluorinated phenylalanines alone and when integrated into proteins exhibit two characteristic and prominent shoulders ("fingers") in the UV spectrum in the range of 260-270 nm, which do not overlap with the contributions of tyrosine and tryptophan residues in the protein UV spectra. Thus, the presence of such "fluorophenylalanine fingers" ("FF fingers") opens a new spectral window to identify the labeled target protein among other nonlabeled cellular proteins in preparative work by simple UV spectroscopy. In the coming era of proteomics such a reliable, cheap, and easy reproducible methodology might have a great potential for speeding up the identification and characterization of target molecules in the total protein output from the genomes of a variety of organisms.

Synthesis of beta-(1-azulenyl)-L-alanine as a potential blue-colored fluorescent tryptophan analog and its use in peptide synthesis.

Acetyl-beta-(1-azulenyl)-D,L-alanine has been synthesized in high overall yield by the malonic ester condensation procedure, and the racemate has been enzymatically resolved with acylase I from Aspergillus melleus. The enantiomerically pure L-amino acid is of interest as a blue-colored fluorescent tryptophan analog. The bioactivity data of a heptagastrin analog containing it suggests that the planar aromatic azulene moiety may indeed mimic the tryptophan side chain to some extent, and the spectral properties of the azulene moiety makes beta-(1-azulenyl)-L-alanine of potential value as a UV and fluorescence probe in synthetic peptides, and possibly even in proteins if bioincorporation succeeds with chemically misacylated tRNAs.

Atomic mutations at the single tryptophan residue of human recombinant annexin V: effects on structure, stability, and activity.

The single tryptophan residue (Trp187) of human recombinant annexin V, containing 320 residues and 5328 atoms, was replaced with three different isosteric analogues where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine. Such single atom exchanges of H --> F represent atomic mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure. These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form. But the mutants exhibit significant differences in stability, folding cooperativity, biological activity, and fluorescence properties if compared to the wild-type protein. These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts. The changes in local geometry that are below resolution of protein X-ray crystallographic studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom. Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochemical properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein molecule.

Toward the experimental codon reassignment in vivo: protein building with an expanded amino acid repertoire.

The high precision and fidelity of the genetic message transmission are ensured by numerous proofreading steps, from DNA replication and transcription to protein translation. The key event for translational fidelity is the proper codon assignment for 20 canonical amino acids. An experimental codon reassignment is possible for noncanonical amino acids in vivo using artificially constructed expression hosts under efficient selective pressure. However, such amino acids may interfere with the cellular metabolism and thus do not belong to the 'first' or 'restricted' part of the universal code, but rather to a second or 'relaxed' part, which is limited mainly by the downstream proofreading in the natural translational machinery. Correspondingly, not all possible alpha-amino acids can be introduced into proteins. The aim of this study is to discuss biological and evolutionary constraints on possible candidates for this second coding level of the universal code. Engineering of such a 'second' code is expected to have great academic as well as practical impact, ranging from protein folding studies to biomedicine.

Atomic mutations in annexin V--thermodynamic studies of isomorphous protein variants.

We have recently developed methods for specific and high-level replacement of methionine with 2-aminohexanoic acid, selenomethionine and telluromethionine as isosteric and atomic analogues for structural investigations of human recombinant annexin V. The variants formed isomorphic crystals and retained the parent three-dimensional fold and bioactivities. Folding parameters were determined from thermal and chemical unfolding to partially denatured states. Stabilities estimated from guanidinium chloride unfolding equilibria are not changed significantly for the atomic mutants (S-->Se-->Te) while the denaturation midpoint is shifted toward lower values with an increase of the m values at the increase of hydrophobicity. In contrast, stabilities in urea are considerably affected by the atomic substitutions, decreasing together with the m and [D]1/2 values. The methylene and selenium variants are identical within the limits of error of all measurements performed here. The physical parameters of the amino acid analogues and the values derived from the slopes of the unfolding data are highly correlated. This approach demonstrates how systematic variations in atomic number at the site of replacement (atomic mutations) can provide a method to probe specific folding properties of proteins.

Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V.

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a non-natural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coli strain by fermentation procedures in minimal medium. Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses. The wild-type protein and its per-Pro(S) mutant were found to crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and per-Pro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced Tm (-4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.

Expression, purification, characterization, and X-ray analysis of selenomethionine 215 variant of leukocyte collagenase.

Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et. al. (1993) FEBS 331, 134-140; Stöcker et al. (1995) Protein Sci. 4, 823-840]. The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80-Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in change kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon area denaturation. The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.

Bioincorporation of telluromethionine into proteins: a promising new approach for X-ray structure analysis of proteins.

A simple and efficient method for the specific and quantitative replacement of the naturally occurring amino acid methionine by its isosteric analogue telluromethionine in the expression of recombinant proteins has been developed. The method requires a controlable and competitive expression system like the bacteriophage T7 polymerase/promoter in a methionine-auxotrophic host. Using methionine-auxotrophic Escherichia coli strains, incorporation of telluromethionine at high yields has been achieved for human recombinant annexin V, human mitochondrial transamidase, Arabidopsis glutathione-S-transferase and the N-terminal domain of Salmonella tailspike adhesion protein as confirmed by amino acid, mass-spectrometric and X-ray analyses. Expressed and purified telluromethionine-proteins and native proteins were found to crystallise isomorphously. In terms of efficient bio-expression, isomorphism of crystals and relative abundance of methionine residues, the production of telluromethionine-proteins as heavy-atom derivatives offers a valid and general approach in X-ray analysis by the method of multiple isomorphous replacement.

Phage P22 tailspike protein: crystal structure of the head-binding domain at 2.3 A, fully refined structure of the endorhamnosidase at 1.56 A resolution, and the molecular basis of O-antigen recognition and cleavage.

The tailspike protein of Salmonella phage P22 is a viral adhesion protein with both receptor binding and destroying activities. It recognises the O-antigenic repeating units of cell surface lipopolysaccharide of serogroup A, B and D1 as receptor, but also inactivates its receptor by endoglycosidase (endorhamnosidase) activity. In the final step of bacteriophage P22 assembly six homotrimeric tailspike molecules are non-covalently attached to the DNA injection apparatus, mediated by their N-terminal, head-binding domains. We report the crystal structure of the head-binding domain of P22 tailspike protein at 2.3 A resolution, solved with a recombinant telluromethionine derivative and non-crystallographic symmetry averaging. The trimeric dome-like structure is formed by two perpendicular beta-sheets of five and three strands, respectively in each subunit and caps a three-helix bundle observed in the structure of the C-terminal receptor binding and cleaving fragment, reported here after full refinement at 1.56 A resolution. In the central part of the receptor binding fragment, three parallel beta-helices of 13 complete turns are associated side-by-side, while the three polypeptide strands merge into a single domain towards their C termini, with close interdigitation at the junction to the beta-helix part. Complex structures with receptor fragments from S. typhimurium, S. enteritidis and S. typhi253Ty determined at 1.8 A resolution are described in detail. Insertions into the beta-helix form the O-antigen binding groove, which also harbours the active site residues Asp392, Asp395 and Glu359. In the intact structure of the tailspike protein, head-binding and receptor-binding parts are probably linked by a flexible hinge whose function may be either to deal with shearing forces on the exposed, 150 A long tailspikes or to allow them to bend during the infection process.

Chalcogen-analogs of amino acids. Their use in X-ray crystallographic and folding studies of peptides and proteins.

Using methionine-auxotrophic Escherichia coli strains quantitative biosynthetic replacement of the methionine residues by seleno- and telluromethionine but not by methoxinine was achieved in various model proteins, clearly indicating a limited tolerance in the editing range of methionyl-tRNA synthetase. For expression of the protein variants the acetyl derivatives of the chalcogen-analogs of methionine, obtained by a new and highly efficient synthetic procedure, proved to be the ideal source in the growth media as they were found to be significantly more stable than the underivatized methionine analogs. The conformational properties in solution, the folding and unfolding parameters as well as X-ray crystallographic data confirmed the highly isomorphous character of the atomic mutants and thus the usefulness of this concept in X-ray analysis of proteins. Quantitative replacement of cysteine residues by selenocysteine has recently been achieved using cysteine-auxotrophic E. coli strains, but a selective replacement of cysteine residues by employing the natural translational machinery of selenocysteine is also conceivable. We have therefore performed a detailed study on synthetic selenocysteine-peptides in order to determine the redox potential of this cysteine analog, and thus the ability of related peptide and protein analogs to undergo the correct oxidative folding. Since the redox potential of selenocysteine was found to be significantly more reducing than that of the parent amino acid, selective formation of a diselenide bridge in presence of additional cysteine residues is highly favored as well documented in the case of the synthetic bis-selenocysteine-endothelin I analog. These results confirm that even cysteine residues may represent an interesting target for the design and expression of isomorphous heteroatomic analogs of proteins.

High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli.

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins.