Pigeon egg white protein-based transparent durable hydrogel via monodisperse ionic surfactant-mediated protein condensation.

The thermal gelation property of proteins is useful in creating protein-based materials. The gelation of protein solution often proceeds by the random aggregation of denatured proteins, and the protein-based gels are typically brittle or opaque, or both. Improvement in the mechanical and optical properties of protein-based materials are required for them to be practical and functional. This study investigated pigeon egg white, which is semitransparent in its thermally gelled state, as a protein source for creating hydrogel materials. The protein thermal gelation process was initiated from the orderly condensed state of proteins complexed with monodisperse ionic surfactants to suppress random aggregation. The resultant gel showed transparency in the visible light region and was not destroyed at 99% compression under 17.8 MPa compressive stress, 350-fold higher than the compressive fracture strength of typical boiled pigeon egg white. These results showed that durable transparent hydrogels could be fabricated by the rational combination of natural proteins and surfactants.

Proteome Analysis of Phase-Separated Condensed Proteins with Ionic Surfactants Revealed Versatile Formation of Artificial Biomolecular Condensates.

The formation of highly condensed, native proteins is important for the development of protein-based drugs and materials. In the cell, various types of liquid droplets with broad functions are formed by the spontaneous condensation of protein, as a physiological response. These droplets lack a surrounding membrane but are phase-separated from the water medium. These types of phase-separated states of proteins have potential applications in biotechnology. Recently, we have developed an artificial phase-separated liquid of condensed native proteins, termed a protein condensate (PC), formed by electrostatic complexation with ionic surfactants. Here we report the applicability of PC formation, studied using an E. coli extract as the protein source. The addition of anionic and cationic surfactants at a specific ratio to the E. coli extract resulted in PC formation. A proteome analysis showed that the PC thus formed contained about 600 kinds of proteins, representing 65% of the uniquely detected proteins and confirming the high versatility of PC formation. A statistical analysis revealed that a variety of types of proteins with a wide range of molecular weights and isoelectric points could form PCs.

Water-Rich Fluid Material Containing Orderly Condensed Proteins.

A fluid material with high protein content (120-310 mg mL-1 ) was formed through the ordered self-assembly of native proteins segregated from water. This material is instantly prepared by the simple mixing of a protein solution with anionic and cationic surfactants. By changing the ratio of the surfactants based on the electrostatic characteristics of the target protein, we observed that the surfactants could function as a versatile molecular glue for protein assembly. Moreover, these protein assemblies could be disassembled back into an aqueous solution depending on the salt conditions. Owing to the water-retaining properties of the hydrophilic part of surfactants, the proteins in this material are in a water-rich environment, which maintains their native structure and function. The inclusion of water also provides functional extensibility to this material, as demonstrated by the preparation of an enzymatically active gel. We anticipate that the unique features of this material will permit the use of proteins not only in solution but also as elements of integrated functionalized materials.

Atelocollagen-templated fabrication of tangled fibrous silica.

Protein-templated structured silica and titania are fabricated via a biomimetic method based on the synergistic effect of amine/carboxyl complexes under ambient conditions. Atelocollagen-templated silica showed a tangled fibrous structure with a smooth surface. The number of carboxyl groups of a protein is an important factor for homogeneous silica growth.

Peptide sequences converting polyglutamine into a prion in yeast.

Amyloids are ordered protein aggregates composed of cross-ß sheet structures. Amyloids include prions, defined as infectious proteins, which are responsible for mammalian transmissible spongiform encephalopathies, and fungal prions. Although the conventional view is that typical amyloids are associated with nontransmissible mammalian neurodegenerative diseases such as Alzheimer's disease, increasing evidence suggests that the boundary between transmissible and nontransmissible amyloids is ambiguous. To clarify the mechanism underlying the difference in transmissibility, we investigated the dynamics and the properties of polyglutamine (polyQ) amyloids in yeast cells, in which the polyQ aggregates are not transmissible but can be converted into transmissible amyloids. We found that polyQ had an increased tendency to form aggregates compared to the yeast prion Sup35. In addition, we screened dozens of peptides that converted the nontransmissible polyQ to transmissible aggregates when they flanked the polyQ stretch, and also investigated their cellular dynamics aiming to understand the mechanism of transmission.

DNA-maleimide: an improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein.

BACKGROUND: Thiol-mediated redox regulation of proteins plays a key role in many cellular processes. METHODS: To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal). RESULTS: DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions. CONCLUSIONS: DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein. GENERAL SIGNIFICANCE: The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.

Flexibility of GroES mobile loop is required for efficient chaperonin function.

Chaperonin GroEL and its partner GroES assist the folding of nascent and stress-damaged proteins in an ATP-dependent manner. Free GroES has a flexible "mobile loop" and binds to GroEL through the residues at the tip of the loop, capping the central cavity of GroEL to provide the substrate polypeptide a cage for secure in-cage folding. Here, we show that restriction of the flexibility of the loop by a disulfide cross-linking between cysteines within the loop results in the inefficient formation of a stable GroEL-polypeptide-GroES ternary complex and inefficient folding. Then, we generated substrate proteins with enhanced binding affinity to GroEL by fusion of one or two SBP (strongly binding peptide for GroEL) sequences and examined the effect of disulfide cross-linking on the assisted folding. The results indicate that the higher the binding affinity of the substrate polypeptide to GroEL, the greater the contribution of the mobile loop flexibility to efficient in-cage folding. It is likely that the flexibility helps GroES capture GroEL's binding sites that are already occupied by the substrate polypeptide with various binding modes.

Nano-scale alignment of proteins on a flexible DNA backbone.

Nano-scale alignment of several proteins with freedom of motion is equivalent to an enormous increase in effective local concentration of proteins and will enable otherwise impossible weak and/or cooperative associations between them or with their ligands. For this purpose, a DNA backbone made of six oligodeoxynucleotide (ODN) chains is designed in which five double-stranded segments are connected by four single-stranded flexible linkers. A desired protein with an introduced cysteine is connected covalently to the 5'-end of azido-ODN by catalyst-free click chemistry. Then, six protein-ODN conjugates are assembled with their complementary nucleotide sequences into a single multi-protein-DNA complex, and six proteins are aligned along the DNA backbone. Flexible alignment of proteins is directly observed by high-speed AFM imaging, and association of proteins with weak interaction is demonstrated by fluorescence resonance energy transfer between aligned proteins.

Probing open conformation of GroEL rings by cross-linking reveals single and double open ring structures of GroEL in ADP and ATP.

Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroEL(OO)), one in ADP (GroEL(O)), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL(OO). The isolated GroEL(O) and GroEL(OO), which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL(OO) and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL.

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.