Comparative Analysis of Aggregation of Thermus thermophilus Ribosomal Protein bS1 and Its Stable Fragment.

Functionally important multidomain bacterial protein bS1 is the largest ribosomal protein of subunit 30S. It interacts with both mRNA and proteins and is prone to aggregation, although this process has not been studied in detail. Here, we obtained bacterial strains overproducing ribosomal bS1 protein from Thermus thermophilus and its stable fragment bS1(49) and purified these proteins. Using fluorescence spectroscopy, dynamic light scattering, and high-performance liquid chromatography combined with mass spectrometric analysis of products of protein limited proteolysis, we demonstrated that disordered regions at the N- and C-termini of bS1 can play a key role in the aggregation of this protein. The truncated fragment bS1(49) was less prone to aggregation compared to the full-size bS1. The revealed properties of the studied proteins can be used to obtain protein crystals for elucidating the structure of the bS1 stable fragment.

The Molten Globule Concept: 45 Years Later.

In this review, we describe traditional systems where the molten globule (MG) state has been detected and give a brief description of the solution of Levinthal's paradox. We discuss new results obtained for MG-mediated folding of "nontraditional" proteins and a possible functional role of the MG. We also report new data on the MG, especially the dry molten globule.

Intermediate States of Apomyoglobin: Are They Parts of the Same Area of Conformations Diagram?

Several research teams have reported detection and characterization of various apomyoglobin intermediate states different in their accumulation mode, thus putting a natural question as to proportions of these intermediates. The current report presents spectral properties of sperm whale apomyoglobin studied over a wide range of conditions with the use of circular dichroism and fluorescence techniques. Based on the experimental data, a diagram of apomyoglobin conformational states has been constructed. It shows that though induced by various denaturants, all the observed intermediates belong to one and the same area in the diagram.

How membrane surface affects protein structure.

The immediate environment of the negatively charged membrane surface is characterized by decreased dielectric constant and pH value. These conditions can be modeled by water-alcohol mixtures at moderately low pH. Several globular proteins were investigated under these conditions, and their conformational behavior in the presence of phospholipid membranes was determined, as well as under conditions modeling the immediate environment of the membrane surface. These proteins underwent conformational transitions from the native to a molten globule-like state. Increased flexibility of the protein structure facilitated protein functioning. Our experimental data allow understanding forces that affect the structure of a protein functioning near the membrane surface (in other words, in the membrane field). Similar conformational states are widely reported in the literature. This indicates that the negatively charged membrane surface can serve as a moderately denaturing agent in the cell. We conclude that the effect of the membrane field on the protein structure must be taken into account.

Apomyoglobin mutants with single point mutations at val10 can form amyloid structures at permissive temperature.

Formation of amyloid-like protein aggregates in human organs and tissues underlies many serious diseases, therefore being in the focus of numerous biochemical, medical, and molecular biological studies. So far, formation of amyloids by globular proteins has been studied mostly under conditions that strongly destabilized their native structure. Here we present our results obtained at permissive temperature by thioflavin T fluorescence, far UV CD, IR spectroscopy, and electron microscopy. We used apomyoglobin and its mutants with Ala or Phe substituted for Val10 that are structurally close to wild type apomyoglobin. It is shown that at permissive temperature the ability of the protein to form amyloids depends on the extent of its structural destabilization, but not on hydrophobicity of the substituting residue. A possible difference between amyloids formed by strongly destabilized proteins and those yielded by proteins with a slightly fluctuating native structure, as well as the stroke and infarction effect on the ability of proteins to form amyloid structures, are discussed.

[Kinetics of interaction between apomyoglobin and phospholipid membrane].

The interaction of apomyoglobin and its mutant forms with phospholipid membranes was studied using tryptophan fluorescence and CD in the far UV-region. It is shown that a negatively charged phospholipid membrane can have a double effect on the structure of protein molecule upon their interaction: it denatures the native structure of the protein to its intermediate state similar to that in solution, acting as a moderately denaturing reagent. On the other hand, it can structure the unfolded protein to the same intermediate state stabilizing its structure. The kinetics of interaction between the protein and its mutant forms and the phospholipid membrane depends on the charge of the membrane surface. Here the rate of this interaction depends on the phospholipids vesicle concentration and the protein molecule stability increasing with a decrease of the latter. The importance of the obtained results for the folding of membrane proteins and the choice of the pathway for target delivery of protein drugs are discussed.

pH-induced equilibrium unfolding of apomyoglobin: substitutions at conserved Trp14 and Met131 and non-conserved Val17 positions.

A number of residues in globins family are well conserved but are not directly involved in the primary oxygen-carrying function of these proteins. A possible role for these conserved, non-functional residues has been suggested in promoting a rapid and correct folding process to the native tertiary structure. To test this hypothesis, we have studied pH-induced equilibrium unfolding of mutant apomyoglobins with substitutions of the conserved residues Trp14 and Met131, which are not involved in the function of myoglobin, by various amino acids. This allowed estimating their impact on the stability of various conformational states of the proteins and selecting conditions for a folding kinetics study. The results obtained from circular dichroism, tryptophan fluorescence, and differential scanning microcalorimetry for these mutant proteins were compared with those for the wild type protein and for a mutant with the non-conserved Val17 substituted by Ala. In the native folded state, all of the mutant apoproteins have a compact globular structure, but are destabilized in comparison to the wild type protein. The pH-induced denaturation of the mutant proteins occurs through the formation of a molten globule-like intermediate similar to that of the wild type protein. Thermodynamic parameters for all of the proteins were calculated using the three state model. Stability of equilibrium intermediates at pH ~4.0 was shown to be slightly affected by the mutations. Thus, all of the above substitutions influence the stability of the native state of these proteins. The cooperativity of conformational transitions and the exposed to solvent protein surface were also changed, but not for the substitution at Val17.

[Equilibrium unfolding of mutant apomyoglobins with substitutions of conserved nonfunctional residues by alanine].

The problems of protein aggregation and protein misfolding in the cell are connected with the appearance of many genetic diseases. Both processes can be a consequence of substitutions of certain amino acid residues in proteins. The substitutions can influence the protein stability and protein folding rates in both the intermediate and the native states. We have studied equilibrium urea unfolding of mutant forms of apomyoglobin with substitutions of conserved nonfunctional residues by Ala to estimate their influence on protein stability. These residues include Val10, Trp14, Ilel11, Leu115, Met131 and Leu135. Conformational transitions were monitored by intrinsic Trp fluorescence and by circular dichroism spectra in the far UV region. Free energy changes upon the transition from the native to intermediate state and from the intermediate to unfolded state were determined. It was shown that all substitutions used lead to an appreciable decrease of the apomyoglobin native state stability, whereas the stability of the intermediate state is affected substantially smaller.