Lisa M. Gloss

Halophilic enzymes: proteins with a grain of salt.

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.

The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases.

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose.

Folding mechanism of the (H3–H4)2 histone tetramer of the core nucleosome

To further understand oligomeric protein assembly, the folding and unfolding kinetics of the H3–H4 histone tetramer have been examined. The tetramer is the central protein component of the core nucleosome, which is the basic unit of DNA compaction into chromatin in the eukaryotic nucleus. This report provides the first kinetic folding studies of a protein containing the histone fold dimerization motif, a motif observed in several protein–DNA complexes. Previous equilibrium unfolding studies have demonstrated that, under physiological conditions, there is a dynamic equilibrium between the H3–H4 dimer and tetramer species. This equilibrium is shifted predominantly toward the tetramer in the presence of the organic osmolyte trimethylamine‐N‐oxide (TMAO). Stopped‐flow methods, monitoring intrinsic tyrosine fluorescence and far‐UV circular dichroism, have been used to measure folding and unfolding kinetics as a function of guanidinium hydrochloride (GdnHCl) and monomer concentrations, in 0 and 1 M TMAO. The assignment of the kinetic phases was aided by the study of an obligate H3–H4 dimer, using the H3 mutant, C110E, which destabilizes the H3–H3′ hydrophobic four‐helix bundle tetramer interface. The proposed kinetic folding mechanism of the H3–H4 system is a sequential process. Unfolded H3 and H4 monomers associate in a burst phase reaction to form a dimeric intermediate that undergoes a further, first‐order folding process to form the native dimer in the rate‐limiting step of the folding pathway. H3–H4 dimers then rapidly associate with a rate constant of ≥107 M−1sec−1 to establish a dynamic equilibrium between the fully assembled tetramer and folded H3–H4 dimers.

The H2A.Z/H2B dimer is unstable compared to the dimer containing the major H2A isoform

The nucleosome, the basic fundamental repeating unit of chromatin, contains two H2A/H2B dimers and an H3/H4 tetramer. Modulation of the structure and dynamics of the nucleosome is an important regulation mechanism of DNA‐based chemistries in the eukaryotic cell, such as transcription and replication. One means of altering the properties of the nucleosome is by incorporation of histone variants. To provide insights into how histone variants may impact the thermodynamics of the nucleosome, the stability of the heterodimer between the H2A.Z variant and H2B was determined by urea‐induced denaturation, monitored by far‐UV circular dichroism, intrinsic Tyr fluorescence intensity, and anisotropy. In the absence of stabilizing agents, the H2A.Z/H2B dimer is only partially folded. The stabilizing cosolute, trimethylamine‐N‐oxide (TMAO) was used to promote folding of the unstable heterodimer. The equilibrium stability of the H2A.Z/H2B dimer is compared to that of the H2A/H2B dimer. The equilibrium folding of both histone dimers is highly reversible and best described by a two‐state model, with no detectable equilibrium intermediates populated. The free energies of unfolding, in the absence of denaturant, of H2A.Z/H2B and H2A/H2B are 7.3 kcal mol−1 and 15.5 kcal mol−1, respectively, in 1 M TMAO. The H2A.Z/H2B dimer is the least stable histone fold characterized to date, while H2A/H2B appears to be the most stable. It is speculated that this difference in stability may contribute to the different biophysical properties of nucleosomes containing the major H2A and the H2A.Z variant.

Revealing transient structures of nucleosomes as DNA unwinds

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

Kinetic folding of Haloferax volcanii and Escherichia coli dihydrofolate reductases: haloadaptation by unfolded state destabilization at high ionic strength.

Salts affect protein stability by multiple mechanisms (e.g., the Hofmeister effect, preferential hydration, electrostatic effects and weak ion binding). These mechanisms can affect the stability of both the native state and the unfolded state. Previous equilibrium stability studies demonstrated that KCl stabilizes dihydrofolate reductases (DHFRs) from Escherichia coli (ecDHFR, E. coli DHFR) and Haloferax volcanii (hvDHFR1, H. volcanii DHFR encoded by the hdrA gene) with similar efficacies, despite adaptation to disparate physiological ionic strengths (0.2 M versus 2 M). Kinetic studies can provide insights on whether equilibrium effects reflect native state stabilization or unfolded state destabilization. Similar kinetic mechanisms describe the folding of urea-denatured ecDHFR and hvDHFR1: a 5-ms stopped-flow burst-phase species that folds to the native state through two sequential intermediates with relaxation times of 0.1-3 s and 25-100 s. The latter kinetic step is very similar to that observed for the refolding of hvDHFR1 from low ionic strength. The unfolding of hvDHFR1 at low ionic strength is relatively slow, suggesting kinetic stabilization as observed for some thermophilic enzymes. Increased KCl concentrations slow the urea-induced unfolding of ecDHFR and hvDHFR1, but much less than expected from equilibrium studies. Unfolding rates extrapolated to 0 M denaturant, k(unf)(H(2)O), are relatively independent of ionic strength, demonstrating that the KCl-induced stabilization of ecDHFR and hvDHFR1 results predominantly from destabilization of the unfolded state. This supports the hypothesis from previous equilibrium studies that haloadaptation harnesses the effects of elevated salt concentrations on the properties of the aqueous solvent to enhance protein stability.

Mutational Analysis of the Stability of the H2A and H2B Histone Monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively--significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol(-1), respectively; at 10 microM, the sum of the stability of the monomers is approximately 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding-initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations-yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central alpha-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.

Asymmetric unwrapping of nucleosomal DNA propagates asymmetric opening and dissociation of the histone core.

Significance Nucleosomes are fundamental protein–DNA structures through which eukaryotes package and organize DNA inside the nucleus. Nucleosomes are disassembled to gain access to the critical information stored in DNA. Here, we describe a new experimental approach that characterizes the kinetics of nucleosome disassembly and the synergy between DNA conformation and protein components. Using NaCl to disrupt electrostatic interactions, we identify kinetic pathways and transient intermediates that reveal how DNA unwrapping and protein dissociation are linked in this macromolecular complex. These dynamic structures may provide new insight into the regulation of DNA access during transcription, replication, and repair. The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A–H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes.

The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A‐H2B and H3‐H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain‐swapped structure comprised of two fused helix:β‐loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid‐like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far‐UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease‐resistant core of the fibrils, with little or no protease protection of the poorly structured N‐terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co‐fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α‐synuclein‐mediated neurotoxicity and fibrillation is considered.

A basic domain in the histone H2B N-terminal tail is important for nucleosome assembly by FACT

Nucleosome assembly in vivo requires assembly factors, such as histone chaperones, to bind to histones and mediate their deposition onto DNA. In yeast, the essential histone chaperone FACT (FAcilitates Chromatin Transcription) functions in nucleosome assembly and H2A–H2B deposition during transcription elongation and DNA replication. Recent studies have identified candidate histone residues that mediate FACT binding to histones, but it is not known which histone residues are important for FACT to deposit histones onto DNA during nucleosome assembly. In this study, we report that the histone H2B repression (HBR) domain within the H2B N-terminal tail is important for histone deposition by FACT. Deletion of the HBR domain causes significant defects in histone occupancy in the yeast genome, particularly at HBR-repressed genes, and a pronounced increase in H2A–H2B dimers that remain bound to FACT in vivo. Moreover, the HBR domain is required for purified FACT to efficiently assemble recombinant nucleosomes in vitro. We propose that the interaction between the highly basic HBR domain and DNA plays an important role in stabilizing the nascent nucleosome during the process of histone H2A–H2B deposition by FACT.