Heat-sterilizable antibody mimics designed on the cold shock protein scaffold from hyperthermophile Thermotoga maritima.

Antibodies and antibody mimics are extensively used in the pharmaceutical industry, where stringent safety standards are required. Implementing heat sterilization during or after the manufacturing process could help prevent contamination by viruses and bacteria. However, conventional antibodies and antibody mimics are not suitable for heat sterilization because they irreversibly denature at high temperatures.

Secretory expression in Bacillus subtilis, purification, and characterization of a persistent protein-degrading enzyme from Nocardiopsis sp. TOA-1.

Keratinase from Nocardiopsis sp. TOA-1 (NAPase) holds significant potential for industrial and medical applications. Here, we developed a heterologous secretory expression system for NAPase in Bacillus subtilis.

Utilization of low-stability variants in protein evolutionary engineering.

Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies.

Conformation state-specific monobodies regulate the functions of flexible proteins through conformation trapping.

Synthetic binding proteins have emerged as modulators of protein functions through protein-protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic-binders-based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type-III domain-based synthetic binding proteins) in regulating the functions of proteins that undergo tens-of-angstroms-scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adk ; a phosphoryl transfer-catalyzing enzyme with a conformational change between OPEN/CLOSED forms).

Induced Circular Dichroism Analysis of Thermally Induced Conformational Changes on Protein Binding Sites Under a Crowding Environment.

Protein-ligand interactions in crowded cellular environments play a crucial role in biological functions. The crowded environment can perturb the overall protein structure and local conformation, thereby influencing the binding pathway of protein-ligand reactions within the cellular milieu. Therefore, a detailed understanding of the local conformation is crucial for elucidating the intricacies of protein-ligand interactions in crowded cellular environments.

Structures of a FtsZ single protofilament and a double-helical tube in complex with a monobody.

FtsZ polymerizes into protofilaments to form the Z-ring that acts as a scaffold for accessory proteins during cell division. Structures of FtsZ have been previously solved, but detailed mechanistic insights are lacking. Here, we determine the cryoEM structure of a single protofilament of FtsZ from Klebsiella pneumoniae (KpFtsZ) in a polymerization-preferred conformation.

An efficient selenium transport pathway of selenoprotein P utilizing a high-affinity ApoER2 receptor variant and being independent of selenocysteine lyase.

Selenoprotein P (SeP, encoded by the SELENOP gene) is a plasma protein that contains selenium in the form of selenocysteine residues (Sec, a cysteine analog containing selenium instead of sulfur). SeP functions for the transport of selenium to specific tissues in a receptor-dependent manner. Apolipoprotein E receptor 2 (ApoER2) has been identified as a SeP receptor.

Dispersion Effect of Molecular Crowding on Ligand-Protein Surface Binding Sites of RNase HI.

The molecular crowding effect on ligand-protein interactions, which plays several crucial roles in life processes, has been investigated using various models by adding crowding agents to mimic the intracellular environment. Several studies evaluating this effect have focused on the ligand-protein binding reaction of well-structured binding sites with rigid conformations. However, the crowding effect on flexible binding sites is not well-understood, especially in terms of the conformations.

Structure-function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis.

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear.

Dynamic Assembly/Disassembly of FtsZ Visualized by High-Speed Atomic Force Microscopy.

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments.

Revisiting the Rate-Limiting Step of the ANS-Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined.

Exploring mutable conserved sites and fatal non-conserved sites by random mutation of esterase from Sulfolobus tokodaii and subtilisin from Thermococcus kodakarensis.

Homologous proteins differ in their amino acid sequences at several positions. Generally, conserved sites are recognized as not suitable for amino acid substitution, and thus in evolutionary protein engineering, non-conserved sites are often selected as mutation sites. However, there have also been reports of possible mutations in conserved sites.

Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures.

The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III.

The effect of salt on the electrostatic interaction of a protein is an important issue, because addition of salt affects protein stability and association/aggregation. Although adding salt is a generally recognized strategy to improve protein stability, this improvement does not necessarily occur. The lack of an effect upon the addition of salt was previously confirmed for the tenth fibronectin type III domain from human fibronectin (FN3) by thermal stability analysis.

Microflow system promotes acetaminophen crystal nucleation.

It is known that interfaces have various impacts on crystallization from a solution. Here, we describe crystallization of acetaminophen using a microflow channel, in which two liquids meet and form a liquid-liquid interface due to laminar flow, resulting in uniform mixing of solvents on the molecular scale. In the anti-solvent method, the microflow mixing promoted the crystallization more than bulk mixing.

Highly active enzymes produced by directed evolution with stability-based selection.

In a directed evolution aimed at improving enzymatic activity, a situation occurs where highly active variants can no longer be obtained from a template protein because the template is already located at a peak (local maximum) in the fitness landscape of activity for the sequence space. To overcome this situation, the template needs to descend the mountain (lose activity) once and climb another higher mountain. However, there is no solid guideline of how the template should go down.

Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C Plant-like High Catalytic Activity.

Photosynthetic rate at the present atmospheric condition is limited by the CO-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (k) and poor affinity for CO (K) and specificity for CO (S). Rubisco in C plants generally shows higher k than that in C plants. Rubisco consists of eight large subunits and eight small subunits (RbcS).

Crystal structure of a GH1 β-glucosidase from Hamamotoa singularis.

A GH1 β-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.

Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli.

FtsZ, a tubulin-like GTPase, is essential for bacterial cell division. In the presence of GTP, FtsZ polymerizes into filamentous structures, which are key to generating force in cell division. However, the structural basis for the molecular mechanism underlying FtsZ function remains to be elucidated.

Spectroscopic Signature of the Steric Strains in an RNase HI Cavity-Filling Destabilized Mutant Protein.

A cavity-filling mutation at a hydrophobic cavity is a useful method for increasing protein stability. This method, however, sometimes destabilizes the protein because of the accompanying structural changes by the steric hindrance around the cavity. Thus, detailed knowledge of unfavorable structural changes is important for a comprehensive understanding of the cavity-filling mutation.

Activity-stability trade-off in random mutant proteins.

In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense.

Monobody-Mediated Alteration of Lipase Substrate Specificity.

Controlling the catalytic properties of enzymes remain an important challenge in chemistry and biotechnology. We have recently established a strategy for altering enzyme specificity in which the addition of proxy monobodies, synthetic binding proteins, modulates the specificity of an otherwise unmodified enzyme. Here, in order to examine its broader applicability, we employed the strategy on Candida rugosa lipase 1 (CRL1), an enzyme with a tunnel-like substrate binding site.

Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain.

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available.

Monobody-mediated alteration of enzyme specificity.

Current methods for engineering enzymes modify enzymes themselves and require a detailed mechanistic understanding or a high-throughput assay. Here, we describe a new approach where catalytic properties are modulated with synthetic binding proteins, termed monobodies, directed to an unmodified enzyme. Using the example of a β-galactosidase from Bacillus circulans, we efficiently identified monobodies that restricted its substrates for its transgalactosylation reaction and selectively enhanced the production of small oligosaccharide prebiotics.