Friedrich von der Haar

Purification of proteins by fractional interfacial salting out on unsubstituted agarose gels.

Abstract Proteins can be precipitated onto the surface of unsubstituted agarose at an ammonium sulfate concentration about 10% lower than needed for precipitation out of solution. The protein is fractionally redissolved by developing agarose columns with a linear, decreasing gradient of ammonium sulfate. The method is characterized by high reproducibility, good purification factors and high recovery of enzymatic activity. As an example the method is applied to the purification of aminoacyl-tRNA synthetases (E.C. 6.1.1.-) specific for phenylalanine, isoleucine and valine.

Über das aminoacylierungsverhalten chemisch modifizierter phenylalaninspezifischer transfer-ribonucleinsäure aus hefe: (1) Glykolspaltung und reduktion zum diol an der 3′-terminalen ribose

The terminal cis‐glycol‐group of phenylalanine specific tRNA (tRNAPhe, I) from yeast was oxidized to the dialdehyde by NaIO4 (tRNAPhe oxi, II) and subsequently reduced to the diol by NaBH4 (tRNAPhe oxi‐red, III). In charging experiments it could be shown that tRNAPhe oxi‐red is still completely specific for phenylalanine. The Michaelis constant of tRNAPhe oxi‐red remains unchanged when compared with tRNAPhe, while the maximun velocity of charging υmax of tRNAPhe oxi‐red is decreased to about one half.

Isoleucyl-tRNA synthetase from baker's yeast: The 3′-hydroxyl group of the 3′-terminal ribose is essential for preventing misacylation of tRNAIle-C-C-A with misactivated valine

It is a well established fact that several aminoacyltRNA synthetases are able to form an (aminoacylAMP-enzyme) complex with non-substrate aminoacids structurally structurally related to the substrate aminoacid (for references see [I]). One of the best documented cases is isoleucyl-tRNA synthetase of Escherichia coli, which is able to activate valine yielding Val-AMP* isoleucyl-tRNA synthetase [2]. This complex is immediately hydrolysed by the addition of tRNA”C-C-A [3,4], indicating that a correction mechanism prevents misacylation of tRNAne-C-C-A. A prerequisite of this correction mechanism is the presence of an intact 3’-adenosine on tRNA@-, since any tRNAUe modified at the 3’end including Be-tRNAIe lacks the ability to hydrolyse the Val-AMPisoleucyl-tRNA synthetase complex [4]. For isoleucyl-tRNA synthetase from Bacillus stearothermophilus it is reported that at temperatures >7O”C the enzyme is able to transfer misactivated valine to tRNAJe-C-C-A [5]. Isoleucyl-tRNA synthetase from baker’s yeast shows almost the same behaviour as the enzyme from E coli with respect to misactivation of valine (unpublished results from this laboratory). In an earlier report [6] we showed that the accepting hydroxyl group for the isoleucyl residue is the 2’. and not the 3’-hydroxyl group of the 3’-terminal adenosine of tRNA. This could be tested by substituting the 3’-adenosine of tRNA*le-C-C-A (A) with 3’deoxyadenosine (B) and 2’-deoxyadenosine (C), respectively [ 61.

In vitro incorporation of 2′-deoxyadenosine and 3′-deoxyadenosine into yeast tRNAPhe using tRNA nucleotidyl transferase, and properties of tRNAPhe-C-C-2′dA and tRNAPhe-C-C-3′dA

Abstract 2′-Deoxyadenosine and 3′-deoxyadenosine (cordycepin) can be incorporated into the 3′-terminal position of tRNA Phe by tRNA nucleotidyl transferase. tRNA Phe -C-C-2′dA and tRNA Phe -C-C-3′dA, missing the cis-diol group at the 3′-terminal end are resistant to periodate oxidation and are not able to form borate complexes. In aminoacylation experiments only the tRNA Phe -C-C-3′dA proved to be chargeable.

NMR study on the methyl and methylene proton resonances of tRNAyeastPhe

Summary The proton resonances of 13 methyl groups and 4 methylene groups belonging to 12 modified bases in tRNAyeastPhe were investigated by 220 MHz NMR spectrometry. The chemical shifts and the linewidths at half height of these assigned resonances in tRNA were measured as a function of temperature from 21° to 80°C. The results indicate: (1) The anticodon loop does not associate with other components of the molecule and the side chain of the Y base protrudes out into the solvent; (2) The methyl groups m5C40,49, m2G10, and m1A58 are not near any diamagnetic regions in the native tRNA; (3) The methyl and methylene groups in m22G, T, and hU are magnetically shielded and immobilized to a great extent in the native conformation, implying that these bases are deeply involved in the tertiary structure of tRNA.

[19] Purification of aminoacyl-tRNA synthetases

Publisher Summary The chapter describes a procedure that allows the purification of several aminoacyl-tRNA synthetases simultaneously to such an extent that each individual can be obtained in a homogeneous form by only one further step. The work described is performed mainly with bakers yeast. It is also successfully applied, with only modifications in the method of cell rupture, to the purification of aminoacyl-tRNA synthetases from Neurospora crassa as well as from several bacteria. The strategy of the procedure described depends on the conditions prepurification of aminoacyl-tRNA synthetases as a group by a series of precipitation steps and adsorption to a cation exchanger. Stepwise elution of subfractions containing only few aminoacyl- tRNA synthetases from the cation exchanger and further purification by salting out on Sepharose 4B, which is applicable to proteins in general. Individual enzymes are obtained in a homogeneous state by only one further step optimized for the individual enzyme.

Crystallization of yeast phenylalanine transfer ribonucleic acid

Abstract Two crystalline forms of yeast phenylalanine transfer RNA have been prepared from water-2-methylpentan-2,4-diol and water-tertiary butanol systems. One of the crystal forms has also been produced by the vapour phase method from water-dioxane and both have been produced by precipitation at a waterbutanol-2 interface. One crystal form is orthorhombic (space group C2221, density 1.47), a = 60.5 A , b = 85 A , c = 234 A and has 32 molecules in the unit cell, i.e. four molecules in the asymmetric unit, and the other is a rhombohedral form (space group R32, density 1.51), a rh = 124 A , α rh = 60.8 ° with 36 molecules in the unit cell, i.e. six molecules in the asymmetric unit. Data were collected to a resolution of 15 A with the orthorhombic crystals and to 7 A with the rhombohedral crystals. The orthorhombic form has a large mosaic spread. The crystals are stable. Yeast phenylalanine transfer RNA recovered from the crystals is chargeable to the same extent as the starting material.

The ligand-induced solubility shift in salting out chromatography: a new affinity technique, demonstrated with phenylalanyl- and isoleucyl-tRNA synthetase from baker's yeast.

Recently I have described the use of salting out chromatography with unsubstituted hydrophilic gels for the purification of proteins [ 1,2], A protein fraction isolated by this procedure is characterised by the identical solubility of all its constituents. A major improvement of this method could be expected if one were able to alter specifically the solubility of one of the components of the mixture, since this single species should, on rechromatography, appear either before or after all the other proteins. Such a slight shift in solubility is expected to occur on complexation of an enzyme with its ligand, and might consequently be the basis for an improvement of salting out chromatography. The usefulness of this principle is demonstrated for phenylalanyl(RC 6.1 .1.20) and isoleucyl-tRNA synthetase (EC 6.1.1.5), which can be subjected to salting out chromatography after complexation with the respective tRNA. Several practical and mechanistic implications are discussed.

Enzyme specificity resulting from proofreading events

In 1974 J. J. Hopfleld developed a mechanistic scheme, called ‘kinetic proofreading’, which was able to explain, at least partially, the high specificity required in biosynthetic processes [l] . In this concept, attention was drawn to the advantages gained by successive steps with respect to enzyme specificity. This scheme was almost immediately accepted as conceptually important, since such an explanation was particularly necessary for enzyme catalysed reactions selecting one substrate from a group of very similar metabolites such as amino acids, aminoacylated tRNA and nucleotides [2]. It was obvious that the differences in the intrinsic free energy of complex formation between an enzyme and two ;uch similar metabolites may not always be large enough o account for the very high specificity required. Let us consider one example. It is known that isoleucyl-tRNA synthetase [E1le] from various sources is able to misactivate valine (Scheme 1) [3,4] .

Mechanism of Aminoacyl-tRNA Synthetases: Recognition and Proofreading Processes

Over a decade has elapsed since it became clear that tRNA is specifically aminoacylated by the cognate aminoacyl-tRNA synthetase to provide the correct aminoacyl-tRNA as a building block for protein biosynthesis (Loftfield 1972; Goddard 1978). Despite many attempts, both theoretical and practical, to understand the pathways leading to the final, error-free product formation, there have been until recently only a few experimental facts with which to approach this all-important aspect of aminoacyl-tRNA synthetase function. The problem is conveniently divisible into two parts. Concerning the small substrate, the amino acid, structural differences may be envisaged as playing a part in the recognition. On the other hand, with regard to tRNA recognition, initial studies were aimed at determining structural differences within the group of ligands and were, perhaps with hindsight, predictably unsuccessful in the search for a recognition region unique to each tRNA. In recent years, partly by abandoning preconceived ideas and recognition theories, great progress has been made in achieving a clearer, semimolecular picture of the processes involved. These new approaches, with their associated jargon (i.e., proofreading, editing, mopping up, triggering, etc.), have revived the interest in the general field of accuracy with a rapid increase in new data that need to be integrated into the framework of previously known facts. We have recently reviewed the field of aminoacyl-tRNA synthetase specificity up to the beginning of 1977 (Igloi and Cramer 1978). We now discuss new results and interpretations with the hope of obtaining a more up-to-date survey of this important area.