Roza Maria Kamp

Application of high-performance liquid chromatographic techniques to the separation of ribosomal proteins of different organisms

The ribosomal proteins from Escherichia coli, Bacillus stearothermophilus and Methanococcus vannielii were separated by size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC), employing new column materials, different gradient systems, and preparative columns, respectively. The purity of the isolated proteins was analysed by one- and two-dimensional gel electrophoresis and by direct micro-sequencing. The separation of ribosomal proteins could be improved by employing propanol gradients in combination with Vydac reversed-phase columns. From the E. coli ribosome, fifteen S and twenty-three L proteins were isolated in sequencer purity by this method. In addition, ion-exchange HPLC was proven to be useful for isolating ribosomal proteins under native conditions: six S proteins and sixteen L proteins from E. coli could be purified. Some of these proteins were not isolated by the reversed-phase procedures, e.g. proteins L9, L14 and L21.

Protein Structure Analysis

The SCOP database aims to provide a detailed and comprehensive description of the structural and evolutionary relationships between all proteins whose structure is known. Proteins are classified to reflect both structural and evolutionary relatedness. Many levels exist in the hierarchy; the principal levels are family, superfamily and fold Family: Clear evolutionarily relationship Superfamily: Probable common evolutionary origin Fold: Major structural similarity SCOP: Structural Classification of Proteins

Purification of Escherichia coli 50 S ribosomal proteins by high performance liquid chromatography.

The 50 S subunit proteins from the Escherichia coli ribosome were purified by size‐exclusion, ion‐exchange or reversed phase high performance liquid chromatography (HPLC) avoiding any precipitation or desalting procedures during isolation. Best resolution of this complex protein mixture was achieved by reversed phase chromatography on supports with short alkyl chains and C18 hydrocarbon‐bonded phases; 23 out of the 32 proteins from the 50 S subunit were purified as shown by two‐dimensional gel electrophoresis, amino acid analysis and direct micro‐sequencing. Protein recoveries varied between 25 and 84% as determined by amino acid analysis. Ribosomal proteins of other organims can be separated under similar conditions.

Desalting and concentration of proteins in dilute solution using reversed-phase high-performance liquid chromatography

A rapid method for desalting and concentrating dilute protein solutions using short reversed-phase columns (3-4 cm) has been described. The recovery of proteins is usually 90-100%. The method is simple and rapid and allows the desalting and concentration of protein samples simultaneously. A wide variety of proteins in the range up to 80 kDa can be desalted in microgram to milligram amounts, and volumes up to 1 liter can be concentrated to a few milliliters by a single injection.

Proteome and Protein Analysis

Selected papers presented at the MPSA 98 are covering new, sensitive and rapid methods for the analysis of proteins, with special emphasis on the total cell proteins, the proteome. In addition to the experimental details, the advantages and limitations of the methodological approaches are discussed. Topics included are: Protein sequencing analysis, protein and peptide sample preparation, mass spectrometry, NMR, analysis of post-translational modifications, purification of recombinant proteins, protein-protein and protein-DNA interactions, structure prediction, modeling and protein folding, functional implications of protein domains and newly emerging methods for the investigation of the proteome, allowing to analyse the expression of genes.

Primary structure of protein S11 from Escherichia coli ribosomes

Protein Sl 1 has by immune electron microscopy been localized on the head and the large lobe [l] or on the platform [2] of the 30 S subunit. It has been crosslinked to proteins S8, S13 and S21 by bifunctional reagents [3]. The distance between the gravity centers of mass of proteins Sl 1 and S12 has been determined to be 114 A and that between Sl 1 and S15 to be 95 A [4]. However, the location of protein SI 1 within the 30 S subunit as deduced from neutron scattering studies is at present dif~c~t to reconcile with the immune electron microscopical results. Reconstruction tests have identified protein Sl 1 as a fidelity protein since its omission leads to a decreased fidelity of translation [S]. Interestingly, protein Sl 1 was one of the proteins found to be affinity-labeled using a mRNA derivative [6]. In this paper the complete primary structure of protein Sl 1 which consists of 128 amino acids residues and has Mr 13 728 is described. We also show the secondary structure of protein Sl 1 using 4 different prediction proxies, F~~ermore, we present data obtained from computer searching for homologous structures between protein Sl 1 on one hand and ribosomal proteins from Escherichia coli and other organisms on the other hand.

Methods in proteome and protein analysis

Part I Structural Proteomics 1 Helix-helix packing between transmembrane fragments Mar Orzaez, Francisco J. Taberner, Enrique Perez-Paya and Ismael Mingarro 2 Mobility studies in Proteins by 15N Nuclear Magnetic Resonance: Rusticyanin as an example Beatrix Jimenez, Jose Maria Moratal, Mario Piccioli and Antonio Donaire 3 Structure and dynamics of proteins in crowded media: A time-resolved fluorescence polarization study Silvia S. Zorrilla, German Rivas, Maria Pilar Lillo Part II Proteome Analysis 4 Analyses of wheat seed proteome: exploring protein-protein interactions by manipulating genome composition Nazrul Islam and Hisashi Hirano 5 Modification-Specific Proteomic Strategy for Identification of Glycosyl-Phosphatidylinosil Anchored Membrane Proteins Felix Elortza, Leonard J. Foster, Allan Stensballe and Ole Norregaard Jensen Part III Structure-Function Correlations 6 Diocleinae lectins: clues to delineate structure/function correlation Francisca Gallego del Sol, Vania M. Ceccatto, Celso S. Nagano, Frederico B.M.B. Moreno, Alexandre H. Sampaio, Thalles B. Grandgeiro, Benildo S. Canada and Juan J. Calvete 7 The contribution of optical biosensors to the analysis of structure-function relationships in proteins Marc H.V. Van Regenmortel Part IV Protein-Protein Interaction 8 The Use of Protein-Protein Interaction Networks for Genome Wide Protein Function Comparisons and Predictions Christine Brun, Anais Baudot, Alain Guenoche and Bernard Jacq 9 Probing ribosomal proteins, capable of interacting with polyamines Dimitrios L.Kalpaxis, Maria A. Xanplanteri, Iioannis Amarantos, Fotini Leontiadou and Theodora Choli-Papadopoulou 10 Application of Optical Biosensors to Structure-Function Studies on the EGF/EGF Receptor System Edouard C. Nice, Bruno Catimel, Julie A. Rothacker, Nathan Hall, Anthony W. Burgess, Thomas P.J. Garrett, Neil . McKern and Colin W. Ward. 11 The functional Interaction Trap: A novel strategy to study specific protein-protein interactions Alok Sharma, Susumu Antoku and Bruce J. Mayer 12 Analysis of protein-protein interaction in complex biological samples by MALDI TOF MS. Feasility and use of the Intensity-fading (IF-) approach. Josep Villanueva, Oscar Yanes, Enrique Querol, Luis Serrano and Francesc X. Aviles Part V Advanced Technologies 13 Accelerator Mass Spectrometry in Protein Research John S. Vogel, Darren J. Hillegonds, Magnus Palmblad, Patrick G. Grand and Graham Bench 14 The use microcalorimetric techniques to study the structure and function of the transferrin receptor from Neisseria meningitidis Tino Krell and Genevieve Renauld-Mongenie 15 The quantitative advantages of an internal standard in multiplexing 2D electrophoresis John Prime, Andrew Alban, Edward Hawkins and Barry Hughes 16 Genetic engineering of bacterial and eukaryotic ribosomal proteins for investigation on elongation arrest of nascent polypeptides and cell differentiation Fotini Leontiadou, Christina Matragou, Philippos Kotakis, Dimitrios L. Kalpakis, Ioanis Vizirianakis, Sofia Kouidou, Asterios Tsifsoglou and Theodora Choli-Papadopoulou 17 MALDI-MS Analysis of Peptides Modified Photolabile Arylazido Groups William Low, James Kang, Michael DiGriccio, Dean Kirby, Marilyn Perrin and Wolfgang H. Fischer Part VI Protein Sequencing and Amino Acids Analysis 18 A New Edman-Type Reagent for High Sensitive Protein Sequencing Christian Wurzel, Barbara zu Lynar, Christoph Radeke, Ralf Kruger, Michael Karas and Brigitte Wittmann-Liebold 19 Amino acid sequencing of

[38] Ribosomal proteins from archaebacteria: High-performance liquid chromatographic purification for microsequence analysis

Publisher Summary This chapter discusses the application of high-performance liquid chromatography (HPLC) methods to the separation of peptide mixtures from the L12 homolog from the large subunit of M. vannielii ribosomes. Further, different HPLC techniques—namely, size exclusion, reversed-phase, and ion-exchange chromatography—are discussed for their ability to resolve different archaebacterial r-protein mixtures. For the identification of proteins after HPLC fractionation, a gel system suitable for archaebacterial protein mixtures was miniaturized so that particular proteins could easily be identified using only small aliquots of the peaks (nanogram amounts) to conserve valuable materials for other purposes. Yields of proteins were calculated by amino acid analysis using precolumn derivatization with o -phthaldialdehyde and reversed phase HPLC. Using the HPLC methods for archaebacterial protein and peptide separations described in this chapter, it is possible to arrive at new sequence data of proteins that would not be feasible without the advanced methodology. The applied methods are suitable not only to structural investigations on r-proteins but also to studies of other proteins of limited sources— for example, multienzyme complexes, receptor proteins, or protein constituents of other cell organdies.

Separation of Peptides

Peptide mapping is a very useful technique for the characterization of proteins. Various rather simple methods can be employed for the separation of peptides, e.g., fingerprinting on thin-layer sheets or a combination of gel filtration or ion exchange chromatography with one- or two-dimensional thin-layer chromatography.

The primary structure of protein L9 from theEscherichia coliribosome

The primary structure of protein L9 from the large subunit of the E. coli ribosome has completely been determined. Automatic sequencing of the intact protein by means of an improved Beckman sequencer and by manual sequencing of diverse peptides deriving from cleavages with trypsin, thermolysin, chymotrypsin and pepsin by the DABITC/PITC double coupling method [FEBS Lett. (1978) 93, 205–214] have been performed to establish the amino acid sequence. Protein L9 contains 148 amino acid residues and has a molecular mass of 15 696. Predictions of secondary structural elements for this protein have been made. A strong homology exists between the primary structure of the E. coli protein L9 and a B. stearothermophilus ribosomal protein previously crystallized [FEBS Lett. (1979) 103, 66–70].