Mechanoresponsive Protein Crystals for NADH Recycling in Multicycle Enzyme Reactions.

NAD(H)-dependent enzymes play a crucial role in the biosynthesis of pharmaceuticals and fine chemicals, but the limited recyclability of the NAD(H) cofactor hinders its more general application. Here, we report the generation of mechano-responsive PEI-modified Cry3Aa protein crystals and their use for NADH recycling over multiple reaction cycles. For demonstration of its practical utility, a complementary Cry3Aa protein particle containing genetically encoded and co-immobilized formate dehydrogenase for NADH regeneration and leucine dehydrogenase for catalyzing the NADH-dependent l--leucine (l--Leu) biosynthesis has been produced.

A Bioresponsive Genetically Encoded Antimicrobial Crystal for the Oral Treatment of Helicobacter Pylori Infection.

Helicobacter pylori (H. pylori) causes infection in the stomach and is a major factor for gastric carcinogenesis. The application of antimicrobial peptides (AMPs) as an alternative treatment to traditional antibiotics is limited by their facile degradation in the stomach, their poor penetration of the gastric mucosa, and the cost of peptide production.

Pyrrolysine-Inspired in Cellulo Synthesis of an Unnatural Amino Acid for Facile Macrocyclization of Proteins.

Macrocyclization has been touted as an effective strategy to enhance the in vivo stability and efficacy of protein therapeutics. Herein, we describe a scalable and robust system based on the endogenous biosynthesis of a noncanonical amino acid coupled to the pyrrolysine translational machinery for the generation of lasso-grafted proteins. The biosynthesis of the noncanonical amino acid d-Cys-ε-Lys was achieved by hijacking the pyrrolysine biosynthesis pathway, and then, its genetical incorporation into proteins was performed using an optimized PylRS/tRNA pair and cell line.

Insights from the Structure of an Active Form of Cry5B.

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium , has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112-698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27-698) containing this N-terminal extension.

Restoration of p53 activity via intracellular protein delivery sensitizes triple negative breast cancer to anti-PD-1 immunotherapy.

Background: Although immune checkpoint inhibitors (ICIs) have been shown to yield promising therapeutic outcomes in a small subset of patients with triple negative breast cancer (TNBC), the majority of patients either do not respond or subsequently develop resistance. Recent studies have revealed the critical role of gene in cancer immunology. Loss or mutation of p53 in cancer cells has been found to promote their immune escape.

Cytosolic delivery of CDK4/6 inhibitor p16 protein using engineered protein crystals for cancer therapy.

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern.

Efficient encapsulation of functional proteins into erythrocytes by controlled shear-mediated membrane deformation.

Red blood cells (RBCs) are attractive carriers of biomolecular payloads due to their biocompatibility and the ability to shelter their encapsulated cargo. Commonly employed strategies to encapsulate payloads into RBCs, such as hypotonic shock, membrane fusion or electroporation, often suffer from low throughput and unrecoverable membrane impairment. This work describes an investigation of a method to encapsulate protein payloads into RBCs by controlling membrane deformation either transiently or extendedly in a microfluidic channel.

Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis.

We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, , was highly active with IC of 0.

A SUMO1-Derived Peptide Targeting SUMO-Interacting Motif Inhibits α-Synuclein Aggregation.

The accumulation of α-synuclein amyloid fibrils in the brain is linked to Parkinson's disease and other synucleinopathies. The intermediate species in the early aggregation phase of α-synuclein are involved in the emergence of amyloid toxicity and considered to be the most neurotoxic. The N-terminal region flanking the non-amyloid-β component domain of α-synuclein has been implicated in modulating its aggregation.

Enzyme Entrapment in a Protein Crystal.

Cry3Aa is a protein that forms crystals naturally in the bacterium . Here we report that coexpression of Cry3Aa and a lipase without recombinant fusion results in the efficient passive entrapment of the lipase within the nanoporous channels of the resulting crystals. This Cry3Aa crystal-mediated entrapment provides multiple benefits to the lipase including a high enzyme loading, significantly improved thermostability, increased proteolytic resistance, and the ability to be utilized as a recyclable biodiesel catalyst.

Directed evolution of a genetically encoded immobilized lipase for the efficient production of biodiesel from waste cooking oil.

Background: We have recently developed a one-step, genetically encoded immobilization approach based on fusion of a target enzyme to the self-crystallizing protein Cry3Aa, followed by direct production and isolation of the fusion crystals from . Using this approach, lipase A was genetically fused to Cry3Aa to produce a Cry3Aa-lipA catalyst capable of the facile conversion of coconut oil into biodiesel over 10 reaction cycles. Here, we investigate the fusion of another lipase to Cry3Aa with the goal of producing a catalyst suitable for the conversion of waste cooking oil into biodiesel.

Direct production of a genetically-encoded immobilized biodiesel catalyst.

The use of immobilized enzymes as biocatalysts has great potential to improve the efficiency and environmental sustainability of many industrial processes. Here, we report a novel approach that allows for the direct production of a highly active immobilized lipase within the bacterium Bacillus thuringiensis. Cry3Aa-lipA crystals were generated by genetically fusing Bacillus subtilis lipase A to Cry3Aa, a protein that naturally forms crystals in the bacteria.

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

Background: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage.

Cry protein crystals: a novel platform for protein delivery.

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications.

Pyrrolysine-inspired protein cyclization.

The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of "site-specific" non-canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole-like topology. As a proof of concept, we fused the well-studied RGD peptide to the C terminus of an mCherry reporter protein.

A click-and-release pyrrolysine analogue.

What's the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites.

Insight into the substrate length restriction of M32 carboxypeptidases: characterization of two distinct subfamilies.

M32 carboxypeptidases are a distinct family of HEXXH metalloproteases whose structures exhibit a narrow substrate groove that is blocked at one end. Structural alignments with other HEXXH metalloprotease-peptide complexes suggested an orientation in which the substrate is directed towards the back of the groove. This led us to hypothesize, and subsequently confirm that the maximum substrate length for M32 carboxypeptidases is restricted.

SIB-BLAST: a web server for improved delineation of true and false positives in PSI-BLAST searches.

A SIB-BLAST web server (http://sib-blast.osc.edu) has been established for investigators to use the SimpleIsBeautiful (SIB) algorithm for sequence-based homology detection.

A pyrrolysine analogue for protein click chemistry.

Ignoring the STOP sign: A pyrrolysine analogue bearing a terminal alkyne was site-specifically incorporated into recombinant calmodulin (CaM) through a UAG codon. The resulting protein was labeled with an azide-containing dye using a copper(I)-catalyzed click reaction. Subsequent application of an orthogonal cysteine tagging method yielded a CaM labeled with two distinct fluorophores that enabled its study by FRET spectroscopy.

Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase.

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis.

Simple is beautiful: a straightforward approach to improve the delineation of true and false positives in PSI-BLAST searches.

Motivation: The deluge of biological information from different genomic initiatives and the rapid advancement in biotechnologies have made bioinformatics tools an integral part of modern biology. Among the widely used sequence alignment tools, BLAST and PSI-BLAST are arguably the most popular. PSI-BLAST, which uses an iterative profile position specific score matrix (PSSM)-based search strategy, is more sensitive than BLAST in detecting weak homologies, thus making it suitable for remote homolog detection.

Distant homology detection using a LEngth and STructure-based sequence Alignment Tool (LESTAT).

A new machine learning algorithm, LESTAT (LEngth and STructure-based sequence Alignment Tool) has been developed for detecting protein homologs having low-sequence identity. LESTAT is an iterative profile-based method that runs without reliance on a predefined library and incorporates several novel features that enhance its ability to identify remote sequences. To overcome the inherent bias associated with a single starting model, LESTAT utilizes three structural homologs to create a profile consisting of structurally conserved positions and block separation distances.