Optical Characteristics of All Individual Proteins from the Small Subunit of Escherichia coli Ribosomes

Abstract

The procedure of isolation and renaturation of all ribosomal proteins from the 30-S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near-ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins.Circular dichroic spectra of 21 individual proteins from the 30-S subunit of E. coli ribosomes were measured in the range 284-310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190-240 nm. Almost all proteins (except proteins S12, S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near-ultraviolet region (240 -310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied.Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far-ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins ; these characteristics may be useful for comparison of protein preparations obtained by different methods in different laboratories. Ribosomal proteins, as a rule, have no actually known physiological functions in the isolated state. Therefore the concept of the native origin of these proteins can be defined only by the identity of their structure and properties in the isolated state to those within the ribosome. The assembly of active ribosomal subunits from the individual isolated proteins cannot prove their native existence as proteins are used in excess during the assembly; they can renature in the process of assembly and the number of active subunits formed is not Therefore from general considerations it is possible to assume that a smaller radius of gyration [6], a more folded tertiary structure (according to NMR data) [7 -91, a higher cooperativity at heat denaturation [9] and a larger content of ordered secondary structure (secondary folding) [I 01 indicate a greater structural identity to the native state of ribosomal proteins.In the study reported here, circular dichroic (CD) spectra of all individual proteins from the small subunit of Escherichia Abbreviations. RNP, ribonucleoprotein; CD, circular dichroism 100 %.coli ribosomes isolated by LiCl/urea extraction and then transferred to non-denaturing conditions were measured in the range 184-310 nm. Of course, we cannot be absolutely certain that for all the proteins isolated under denaturing conditions, a complete renaturation was achieved ; however, we consider that the CD spectra presented are optical characteristics of the individual ribosom...

A Comparison of Structural and Evolutionary Attributes of Escherichia coli and Thermus thermophilus Small Ribosomal Subunits: Signatures of Thermal Adaptation

“…Both thermophilic and mesophilic S4 protein shows high evolutionary conservation at the O2O regions, compared to D2O regions. S12 is a special case, which is completely disordered in its free cytosolic state (according to experimental results [43]). This might be the reason that no significant difference of evolutionary conservation is observed between its predicted disordered and ordered regions (or D2O and O2O transition regions).…”

Y-Box Binding Proteins in mRNP Assembly, Translation, and Stability Control

“…Of note, many RNA-binding proteins, when unbound, are mostly disordered [21][22][23]. For example, ribosomal proteins and proteins of RNA-containing viruses [24,25] acquire their structure upon RNA binding. Similarly, YB proteins might become structurally arranged when binding to RNA or protein partners; yet so far, the literature offers no such information.…”

Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome

“…D-uptake >50% represents highly flexible/loose conformations, <50% indicates tightly folded structures or/and multimers. A collection of control proteins with different properties revealed the ability of the 2D plot to resolve different folding states: the cytoplasmic IDP RS12 (Venyaminov and Gogia, 1982) mapped to the upper left (red; >80% D-uptake; 200/222 nm ratio>1), while the cytoplasmic folders PpiB (Ikura et al, 2000), PurE (Mathews et al, 1999), and YigZ (Park et al, 2004) to the bottom right corner (blue; <50% D-uptake; 200/222 nm ratio 1 to À1). The partially molten globule and flexible YebF (Prehna et al, 2012), mapped distinctly from folders and IDPs, to the lower middle region, as it combines acquired secondary structure with loose 3D packing (>50% D-uptake; 200/222 nm ratio 1 to À1).…”