Non-isothermal cold crystallization of liquid crystalline porphyrins.

A series of liquid crystalline porphyrins was synthesized, purified, and characterized. Differential scanning calorimetry (DSC) and hot-stage polarized optical microscopy (HS-POM) revealed that the porphyrins in the series with shorter alkyl arm lengths exhibit kinetic cold crystallization, wherein the molecules spontaneously organize into large, disc-like structures that remain stable upon cooling. Using DSC, the kinetic and thermodynamic parameters related to these materials were determined. Analysis of non-isothermal crystallization revealed the presence of multiple nucleation and growth processes related to cold crystallization.

Domain Fusion of Two Oxygenases Affords Organophosphonate Degradation in Pathogenic Fungi.

Proteins of the HD-domain superfamily employ a conserved histidine-aspartate (HD) dyad to coordinate diverse metallocofactors. While most known HD-domain proteins are phosphohydrolases, new additions to this superfamily have emerged such as oxygenases and lyases, expanding their functional repertoire. To date, three HD-domain oxygenases have been identified, all of which employ a mixed-valent FeIIFeIII cofactor to activate their substrates and utilize molecular oxygen to afford cleavage of C-C or C-P bonds via a diferric superoxo intermediate. Phylogenetic analysis reveals an uncharacterized multidomain protein in the pathogenic soil fungus Fonsecaea multimorphosa, herein designated PhoF. PhoF consists of an N-terminal FeII/α-ketoglutarate-dependent domain resembling that of PhnY and a C-terminal HD-domain like that of PhnZ. PhnY and PhnZ are part of an organophosphonate degradation pathway in which PhnY hydroxylates 2-aminoethylphosphonic acid, and PhnZ cleaves the C-P bond of the hydroxylated product yielding phosphate and glycine. Employing electron paramagnetic resonance and Mössbauer spectroscopies in tandem with activity assays, we determined that PhoF carries out the O2-dependent degradation of two aminophosphonates, demonstrating an expanded catalytic efficiency with respect to the individual, but mechanistically coupled PhnY and PhnZ. Our results recognize PhoF as a new example of an HD-domain oxygenase and show that domain fusion of an organophosphonate degradation pathway may be a strategy for disease-causing fungi to acquire increased functional versatility, potentially important for their survival.

The HBx protein from hepatitis B virus coordinates a redox-active Fe-S cluster.

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O2-stable [2Fe-2S] cluster form converts to an O2-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction-oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]2+/1+ couple (-520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.

Characterization of Fe-S Clusters in Proteins by MÓ§ssbauer Spectroscopy.

57Fe MÓ§ssbauer spectroscopy is unparalleled in the study of Fe-S cluster-containing proteins because of its unique ability to detect all forms of iron. Enrichment of biological samples with the 57Fe isotope and manipulation of experimental parameters such as temperature and magnetic field allow for elucidation of the number of Fe-S clusters present in a given protein, their nuclearity, oxidation state, geometry, and ligation environment, as well as any transient states relevant to enzyme chemistry. This chapter is arranged in five sections to help navigate an experimentalist to utilize 57Fe MÓ§ssbauer spectroscopy for delineating the role and structure of biological Fe-S clusters. The first section lays out the tools and technical considerations for the preparation of 57Fe-labeled samples. The choice of experimental parameters and their effects on the MÓ§ssbauer spectra are presented in the following two sections. The last two sections provide a theoretical and practical guide on spectral acquisition and analysis relevant to Fe-S centers.

Hepatitis B Virus Oncoprotein HBx Is Not an ATPase.

HBx is the smallest gene product of the Hepatitis B virus (HBV) and an oncogenic stimulus in chronic infections leading to liver disease. HBx interacts and interferes with numerous cellular processes, but its modes of action remain poorly understood. It has been invoked that HBx employs nucleotide hydrolysis to regulate molecular pathways or protein-protein interactions. In the present study, we reinvestigate the (d)NTP hydrolysis of recombinant HBx to explore its potential as a biochemical probe for antiviral studies. For our investigations, we employed existing soluble constructs (i.e., GST-HBx, MBP-HBx) and engineered new fusion proteins (i.e., DsbC-HBx, NusA-HBx), which are shown to serve as better systems for in vitro research. We performed mutational scanning of the computationally predicted NTP-binding domain, which includes residues associated with clinical cases. Steady-state and end-point activity assays, in tandem with mass-spectrometric analyses, reveal that the observed hydrolysis of all alleged HBx substrates, ATP, dATP, and GTP, is contingent on the presence of the GroEL chaperone, which preferentially copurifies as a contaminant with GST-HBx and MBP-HBx. Collectively, our findings provide new technical standards for recombinant HBx studies and reveal that nucleotide hydrolysis is not an operant mechanism by which HBx contributes to viral HBV carcinogenesis.

The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries.

The histidine-aspartate (HD)-domain protein superfamily contains metalloproteins that share common structural features but catalyze vastly different reactions ranging from oxygenation to hydrolysis. This chemical diversion is afforded by (i) their ability to coordinate most biologically relevant transition metals in mono-, di-, and trinuclear configurations, (ii) sequence insertions or the addition of supernumerary ligands to their active sites, (iii) auxiliary substrate specificity residues vicinal to the catalytic site, (iv) additional protein domains that allosterically regulate their activities or have catalytic and sensory roles, and (v) their ability to work with protein partners. More than 500 structures of HD-domain proteins are available to date that lay out unique structural features which may be indicative of function. In this respect, we describe the three known classes of HD-domain proteins (hydrolases, oxygenases, and lyases) and identify their apparent traits with the aim to portray differences in the molecular details responsible for their functional divergence and reconcile existing notions that will help assign functions to yet-to-be characterized proteins. The present review collects data that exemplify how nature tinkers with the HD-domain scaffold to afford different chemistries and provides insight into the factors that can selectively modulate catalysis.

Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism.

Mycofactocin is a putative redox cofactor and is classified as a ribosomally synthesized and post-translationally modified peptide (RiPP). Some RiPP natural products, including mycofactocin, rely on a radical S-adenosylmethionine (RS, SAM) protein to modify the precursor peptide. Mycofactocin maturase, MftC, is a unique RS protein that catalyzes the oxidative decarboxylation and C-C bond formation on the precursor peptide MftA. However, the number, chemical nature, and catalytic roles for the MftC [Fe-S] clusters remain unknown. Here, we report that MftC binds a RS [4Fe-4S] cluster and two auxiliary [4Fe-4S] clusters that are required for MftA modification. Furthermore, electron paramagnetic resonance spectra of MftC suggest that SAM and MftA affect the environments of the RS and Aux I cluster, whereas the Aux II cluster is unaffected by the substrates. Lastly, reduction potential assignments of individual [4Fe-4S] clusters by protein film voltammetry show that their potentials are within 100 mV of each other.