Jin Ryoun Kim

Ligand binding and allostery can emerge simultaneously.

A heterotropic allosteric effect involves an effector molecule that is distinct from the substrate or ligand of the protein. How heterotropic allostery originates is an unanswered question. We have previously created several heterotropic allosteric enzymes by recombining the genes for TEM1 β‐lactamase (BLA) and maltose binding protein (MBP) to create BLAs that are positively or negatively regulated by maltose. We show here that one of these engineered enzymes has ∼106 M−1 affinity for Zn2+, a property that neither of the parental proteins possesses. Furthermore, Zn2+ is a negative effector that noncompetitively switches off β‐lactam hydrolysis activity. Mutagenesis experiments indicate that the Zn2+‐binding site does not involve a histidine or a cysteine, which is atypical of natural Zn2+‐binding sites. These studies also implicate helices 1 and 12 of the BLA domain in allosteric signal propagation. These results support a model for the evolution of heterotropic allostery in which effector affinity and allosteric signaling emerge simultaneously.

A Strategy for Designing a Peptide Probe for Detection of β‐Amyloid Oligomers

Aggregation of β‐amyloid (Aβ) is implicated in the pathology of Alzheimers disease. Development of a robust strategy to detect Aβ oligomeric intermediates, which have been identified as significant toxic agents, would be highly beneficial in the screening of drug candidates as well as enhancing our understanding of Aβ oligomerization. Rapid, specific and quantitative detection, currently unavailable, would be highly preferred for accurate and reliable probing of transient Aβ oligomers. Here, we report the development of a novel peptide probe, PG46, based on the nature of Aβ self‐assembly and the conformation‐sensitive fluorescence of the biarsenical dye, FlAsH. PG46 was found to bind to Aβ oligomers and displayed an increase in FlAsH fluorescence upon binding. No such event was observed when PG46 was co‐incubated with Aβ low‐molecular‐weight species or Aβ fibrils. Aβ oligomer detection was fast, and occurred within one hour without any additional sample incubation or preparation. We anticipate that the development of a strategy for detection of amyloid oligomers described in this study will be directly relevant to a host of other amyloidogenic proteins.

Urea modulation of β-amyloid fibril growth: Experimental studies and kinetic models

Aggregation of β‐amyloid (Aβ) into fibrillar deposits is widely believed to initiate a cascade of adverse biological responses associated with Alzheimers disease. Although it was once assumed that the mature fibril was the toxic form of Aβ, recent evidence supports the hypothesis that Aβ oligomers, intermediates in the fibrillogenic pathway, are the dominant toxic species. In this work we used urea to reduce the driving force for Aβ aggregation, in an effort to isolate stable intermediate species. The effect of urea on secondary structure, size distribution, aggregation kinetics, and aggregate morphology was examined. With increasing urea concentration, β‐sheet content and the fraction of aggregated peptide decreased, the average size of aggregates was reduced, and the morphology of aggregates changed from linear to a globular/linear mixture and then to globular. The data were analyzed using a previously published model of Aβ aggregation kinetics. The model and data were consistent with the hypothesis that the globular aggregates were intermediates in the amyloidogenesis pathway rather than alternatively aggregated species. Increasing the urea concentration from 0.4 M to 2 M decreased the rate of filament initiation the most; between 2 M and 4 M urea the largest change was in partitioning between the nonamyloid and amyloid pathways, and between 4 M and 6 M urea, the most significant change was a reduction in the rate of filament elongation.

Targeted Control of Kinetics of β-Amyloid Self-association by Surface Tension-modifying Peptides

Brain tissue from Alzheimers patients contains extracellular senile plaques composed primarily of deposits of fibrillar aggregates of β-amyloid peptide. β-Amyloid aggregation is postulated to be a major factor in the onset of this neurodegenerative disease. Recently proposed is the hypothesis that oligomeric intermediates, rather than fully formed insoluble fibrils, are cytotoxic. Previously, we reported the discovery of peptides that accelerate β-amyloid aggregation yet inhibit toxicity in vitro, in support of this hypothesis. These peptides contain two domains: a recognition element designed to bind to β-amyloid and a disrupting element that alters β-amyloid aggregation kinetics. Here we show that the aggregation rate-enhancing activity of the disrupting element correlates strongly with its ability to increase surface tension of aqueous solutions. Using the Hofmeister series as a guide, we designed a novel peptide with terminal side-chain trimethylammonium groups in the disrupting domain. The derivatized peptide greatly increased solvent surface tension and accelerated β-amyloid aggregation kinetics by severalfold. Equivalent increases in surface tension in the absence of a recognition domain had no effect on β-amyloid aggregation. These results suggest a novel strategy for targeting localized changes in interfacial energy to specific proteins, as a way to selectively alter protein folding, stability, and aggregation.

Enzyme stabilization by domain insertion into a thermophilic protein

Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzymes desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37 degrees C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37 degrees C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

Mechanism of Accelerated Assembly of β-Amyloid Filaments into Fibrils by KLVFFK6

Extracellular senile plaques are a central pathological feature of Alzheimers disease. At the core of these plaques are fibrillar deposits of β-amyloid peptide (Aβ). In vitro, Aβ spontaneously assembles into amyloid fibrils of cross-β sheet structure. Although it was once believed that the fibrils themselves were toxic, more recent data supports the hypothesis that aggregation intermediates, rather than fully formed fibrils, are the most damaging to neuronal tissue. In previously published work, we identified several small peptides that interact with Aβ and increase its aggregation rate while decreasing its toxicity. In this work, we examined in detail the interaction between Aβ and one of these peptides. Using a mathematical model of Aβ aggregation kinetics, we show that the dominant effect of the peptide is to accelerate lateral association of Aβ filaments into fibrils.

Crocetin inhibits beta-amyloid fibrillization and stabilizes beta-amyloid oligomers.

Aggregation of a peptide, beta-amyloid (Aβ), is a hallmark molecular process found in Alzheimers disease (AD). During Aβ aggregation, oligomeric and fibrillar Aβ are formed, and these molecular self-assembly steps are implicated in generation of toxic effects in AD. Crocetin is a natural carotenoid dicarboxyl acid displaying various pharmaceutical effects and may be co-localized with Aβ mediated by human serum albumin. In the study presented here, we examined the effects of crocetin on Aβ aggregation in three different molecular pathways. Our results demonstrate that crocetin inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils. Moreover, crocetin caused stabilization of Aβ oligomers and prevented their conversion into Aβ fibrils. Our study reveals potential pathological and pharmaceutical implication of crocetin in AD and suggests possible application of crocetin for currently limited structural studies on unstable Aβ oligomers.

Frozen Cyclohexane-in-Water Emulsion as a Sacrificial Template for the Synthesis of Multilayered Polyelectrolyte Microcapsules

This paper reports the application of frozen cyclohexane-in-water emulsions as sacrificial templates for the fabrication of hollow microcapsules through layer-by-layer assembly of polyelectrolytes, poly(styrenesulfonate sodium salt), and poly(allylamine hydrochloride). Extraction of the cyclohexane phase from frozen emulsions stabilized with 11 polyelectrolyte layers by compatibilization with 30% v/v ethanol leads to the formation of water-filled microcapsules while preserving the spherical geometry. The majority of microcapsules (>90%) are prepared with intact polyelectrolyte membranes as measured by their deformation induced by osmotic pressure. This work provides a new route for the synthesis of hollow multilayered microcapsules under mild operating conditions.

Stability of a guest protein depends on stability of a host protein in insertional fusion

Insertional fusion between host and guest protein domains has been employed to create multi‐domain protein complexes displaying integrated and coupled functionalities. The effects of insertional fusion on the stability of a guest protein are however rather controversial. In the study described here, we examined whether the stability of inserted TEM1 beta‐lactamase (BLA), as a guest protein, might be affected by the stability of a maltodextrin‐binding protein (MBP), as a host protein. Our results indicate that expression levels and in vitro stability of the BLA domain were significantly higher when inserted into thermophilic MBP from Pyrococcus furiosus (PfMBP) compared to mesophilic MBP from Escherichia coli (EcMBP). Moreover, insertion into PfMBP at selected sites was found to improve thermal stability of the BLA domain without compromise in expression levels and BLA activity. Kinetic stabilization during prolonged thermal denaturation of the BLA domain was not guaranteed by insertion into PfMBP, but rather relied on the insertion sites. Taken together, we provide evidence that (i) the stability of the guest protein depended on the stability of the host protein in insertional fusion and (ii) insertion into PfMBP, at least at selected locations, can serve as a novel method of improving protein thermal stability. Bioeng. 2011; 108:1011–1020.

Recent Approaches Targeting Beta-Amyloid for Therapeutic Intervention of Alzheimer's disease

Alzheimers disease (AD) is a neurodegenerative disorder characterized by neuropathological features comprising amyloid deposits and neuronal losses in brain. In AD, aggregation of a β amyloid peptide (Aβ), produced from proteolytic cleavage of amyloid precursor protein, is believed to be implicated in the pathophysiological cascade leading to neuronal death. Most AD drugs currently available can only alleviate symptoms rather than modify the underlying molecular cause of AD. In this review, we describe and discuss the recent patents issued within the past two years focusing on therapeutic interventions targeting at various Aβ-associated pathological mechanisms of AD. The described therapeutic strategies include 1) reduction of synthesis of Aβ, 2) inhibition of Aβ aggregation, 3) immunotherapeutic/enzymatic clearance of Aβ, 4) targeting other amyloidogenic proteins interacting with Aβ and 5) amelioration of Aβ downstream toxic effects. Important issues to be considered for further improvement of therapeutic efficacy of these approaches are also discussed.