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...

Quite a long time ago, Oleg B. Ptitsyn put forward a hypothesis about the possible functional significance of the molten globule (MG) state for the functioning of proteins. MG is an intermediate between the unfolded and the native state of a protein. Its experimental detection and investigation in a cell are extremely difficult. In the last decades, intensive studies have demonstrated that the MG-like state of some globular proteins arises from either their modifications or interactions with protein partners or other cell components. This review summarizes such reports. In many cases, MG was evidenced to be functionally important. Thus, the MG state is quite common for functional cellular proteins. This supports Ptitsyn’s hypothesis that some globular proteins may switch between two active states, rigid (N) and soft (MG), to work in solution or interact with partners.

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.

Engineering of amyloid structures is one of the new perspective areas of protein engineering. Studying the process of amyloid formation can help find ways to manage it in the interests of medicine and biotechnology. One of the promising candidates for the structural basis of artificial functional amyloid fibrils is albebetin (ABB), an artificial protein engineered under the leadership of O.B. Ptitsyn. Various aspects of the amyloid formation of this protein and some methods for controlling this process are investigated in this paper. Four stages of amyloid fibrils formation by this protein from the first non-fibrillar aggregates to mature fibrils and large micron-sized complexes have been described in detail. Dependence of albebetin amyloids formation on external conditions and some mutations also have been described. The introduction of similar point mutations in the two structurally identical α-β-β motifs of ABB lead to different amiloidogenesis kinetics. The inhibitory effect of a disulfide bond and high pH on amyloid fibrils formation, that can be used to control this process, was shown. The results of this work are a good basis for the further design and use of ABB-based amyloid constructs.

The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins’ aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions.

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.