Reentrant condensation of a multicomponent cola/milk system induced by polyphosphate.

Reentrant condensation (RC) is a protein behavior in which the protein solution shifts between the one- and two-phase state more than twice by increasing a single parameter. Although RC would be a candidate mechanism for the physicochemical design of food additives, no realistic model has been established under diverse contaminants like food materials. Here, we found that a mixture of cola and milk yielded RC. At pH 3.2-3.6, cola induced milk condensation at 30-40%, while lower or higher concentrations of cola did not. Furthermore, we reduced this cola/milk system to two pure components, casein in milk and polyphosphate (polyP) in cola, and investigated the characteristics of casein concentration and zeta potential. This was the first experimental demonstration of RC occurrence in a multicomponent system. The well-characterized cola/milk system would explore both the universal nature of proteins and the industrial application of RC.

Dynamics and interactions of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) are widespread in eukaryotes and participate in a variety of important cellular processes. Numerous studies using state-of-the-art experimental and theoretical methods have advanced our understanding of IDPs and revealed that disordered regions engage in a large repertoire of intra- and intermolecular interactions through their conformational dynamics, thereby regulating many intracellular functions in concert with folded domains. The mechanisms by which IDPs interact with their partners are diverse, depending on their conformational propensities, and include induced fit, conformational selection, and their mixtures. In addition, IDPs are implicated in many diseases, and progress has been made in designing inhibitors of IDP-mediated interactions. Here we review these recent advances with a focus on the dynamics and interactions of IDPs.

Rational peptide design for inhibition of the KIX-MLL interaction.

The kinase-inducible domain interacting (KIX) domain is an integral part of the general transcriptional coactivator CREB-binding protein, and has been associated with leukemia, cancer, and various viral diseases. Hence, the KIX domain has attracted considerable attention in drug discovery and development. Here, we rationally designed a KIX inhibitor using a peptide fragment corresponding to the transactivation domain (TAD) of the transcriptional activator, mixed-lineage leukemia protein (MLL). We performed theoretical saturation mutagenesis using the Rosetta software to search for mutants expected to bind KIX more tightly than the wild-type MLL TAD. Mutant peptides with higher helical propensities were selected for experimental characterization. We found that the T2857W mutant of the MLL TAD peptide had the highest binding affinity for KIX compared to the other 12 peptides designed in this study. Moreover, the peptide had a high inhibitory effect on the KIX-MLL interaction with a half-maximal inhibitory concentration close to the dissociation constant for this interaction. To our knowledge, this peptide has the highest affinity for KIX among all previously reported inhibitors that target the MLL site of KIX. Thus, our approach may be useful for rationally developing helical peptides that inhibit protein-protein interactions implicated in the progression of various diseases.

A comparative study of unpasteurized and pasteurized frozen whole hen eggs using size-exclusion chromatography and small-angle X-ray scattering.

Hen eggs are rich in proteins and are an important source of protein for humans. Pasteurized frozen whole hen eggs are widely used in cooking and confectionery and can be stored for long periods. However, processed eggs differ from raw eggs in properties such as viscosity, foaming ability, and thermal aggregation. To develop pasteurized frozen whole egg products with properties similar to those of unpasteurized whole eggs, it is necessary to establish a method that can differentiate between the two egg types with respect to the structures of their proteins. In this study, size-exclusion chromatography (SEC) and SEC coupled with small-angle X-ray scattering (SEC-SAXS) were successfully used to differentiate between the proteins in unpasteurized and pasteurized frozen whole eggs. We found that proteins in the plasma fraction of egg yolk, especially apovitellenins I and II, formed large aggregates in the pasteurized eggs, indicating that their structures are sensitive to temperature changes during pasteurization, freezing, and thawing. The results suggest that SEC and SEC-SAXS can be used to differentiate between unpasteurized and pasteurized frozen whole eggs. Additionally, they may be useful in determining molecular sizes and shapes of multiple components in various complex biological systems such as whole eggs.

Intron-Encoded Domain of Herstatin, An Autoinhibitor of Human Epidermal Growth Factor Receptors, Is Intrinsically Disordered.

Human epidermal growth factor receptors (HER/ERBB) form dimers that promote cell proliferation, migration, and differentiation, but overexpression of HER proteins results in cancer. Consequently, inhibitors of HER dimerization may function as effective antitumor drugs. An alternatively spliced variant of HER2, called herstatin, is an autoinhibitor of HER proteins, and the intron 8-encoded 79-residue domain of herstatin, called Int8, binds HER family receptors even in isolation. However, the structure of Int8 remains poorly understood. Here, we revealed by circular dichroism, NMR, small-angle X-ray scattering, and structure prediction that isolated Int8 is largely disordered but has a residual helical structure. The radius of gyration of Int8 was almost the same as that of fully unfolded states, although the conformational ensemble of Int8 was less flexible than random coils. These results demonstrate that Int8 is intrinsically disordered. Thus, Int8 is an interesting example of an intrinsically disordered region with tumor-suppressive activity encoded by an intron. Furthermore, we show that the R371I mutant of Int8, which is defective in binding to HER2, is prone to aggregation, providing a rationale for the loss of function.

Rational design of a helical peptide inhibitor targeting c-Myb-KIX interaction.

The transcription factor c-Myb promotes the proliferation of hematopoietic cells by interacting with the KIX domain of CREB-binding protein; however, its aberrant expression causes leukemia. Therefore, inhibitors of the c-Myb-KIX interaction are potentially useful as antitumor drugs. Since the intrinsically disordered transactivation domain (TAD) of c-Myb binds KIX via a conformational selection mechanism where helix formation precedes binding, stabilizing the helical structure of c-Myb TAD is expected to increase the KIX-binding affinity. Here, to develop an inhibitor of the c-Myb-KIX interaction, we designed mutants of the c-Myb TAD peptide fragment where the helical structure is stabilized, based on theoretical predictions using AGADIR. Three of the four initially designed peptides each had a different Lys-to-Arg substitution on the helix surface opposite the KIX-binding interface. Furthermore, the triple mutant with three Lys-to-Arg substitutions, named RRR, showed a high helical propensity and achieved three-fold higher affinity to KIX than the wild-type TAD with a dissociation constant of 80 nM. Moreover, the RRR inhibitor efficiently competed out the c-Myb-KIX interaction. These results suggest that stabilizing the helical structure based on theoretical predictions, especially by conservative Lys-to-Arg substitutions, is a simple and useful strategy for designing helical peptide inhibitors of protein-protein interactions.