The effect of a high mobility group protein (HMG 17) on the structure of acetylated and control core HeLa cell chromatin

The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins (Reczek, P.R., Weissman, D., Huvos, P.E. and Fasman, G.D. (1982) Biochemistry 21, 993–1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in T_m, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.     

Isolation of oligonucleosomes from active chromatin using HMG17-specific monoclonal antibodies.

We report the preparation of HMG17-containing oligonucleosomes from chicken embryos and from liver and oviduct of laying hens. Monoclonal antibodies against HMG17 were used for their isolation. An unusual size distribution with respect to their repeat number was observed. The oligonucleosomes of repeat number up to N6 were highly enriched for DNA of the vitellogenin II gene (liver) and for DNA of the ovalbumin and lysozyme genes (oviduct).     

Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells.

The nonhistone chromosomal protein HMG1 associated rapidly with the nuclei of HeLa cells and bovine fibroblasts following its introduction into the cytoplasm by red cell-mediated microinjection. A number of non-nuclear proteins, on the other hand, failed to concentrate in HeLa or bovine fibroblast nuclei. Autoradiography of thin sections showed that 125I-labeled HMG1 localized within nuclei, and further established that it remained associated with metaphase chromosomes at mitosis. When uninjected HeLa cells were fused with 125I-HMG1-injected HeLa cells, the labeled molecules equilibrated between nuclei within 12 hr. Similar results were obtained with bovine fibroblasts, indicating that a dynamic equilibrium exists between HMG1 and chromatin within living cells. Electrophoresis of 125I-HMG1 retrieved from HeLa cells or bovine fibroblasts up to 48 hr after injection showed that more than 80% of the molecules were intact. Autoradiographic analysis of cells fixed over a period of several days after injection produced apparent half-lives for 125I-HMG1 of 80 hr in HeLa cells and 100 hr in bovine fibroblasts.     

Differences between some properties of acetylated and nonacetylated forms of HMG1 protein

There are data suggesting that HMG1 protein may be involved in DNA replication. Recently we have found that only the acetylated form of the protein generates tetramers, stimulates the activity of DNA polymerase alpha (EC 2.7.7.7) (with activated DNA as a template) and forms a specific complex with it. This paper compares some properties of the acetylated and nonacetylated forms of HMG1 protein and shows that it is only the acetylated form which serves as a histone assembly factor, increases the melting temperature of poly d[(A-T)] and stimulates the activity of DNA polymerase alpha when histone H1-depleted chromatin is used as a template.     

Effect of sorbed water on the thermal stability of soybean protein

A soybean protein isolate (SPI), and its beta-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS-PAGE of the beta-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 degrees C showed that the amount of the monolayer adsorption of these preparations was about 6-9%, the value for the beta-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The beta-conglycinin and glycinin preparations were respectively denatured at around 75 degrees C and 86 degrees C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.     

Conformation of control and acetylated HeLa stripped chromatin after reassociation with H1

The effect of histone H1 on the conformation and stability of control and acetylated HeLa high-molecular-weight chromatin that had been stripped of H1 and nonhistone proteins was compared by circular dichroism (CD) and thermal denaturation measurements. Two different preparations of H1, originating from rat thymus and chicken erythrocyte, were used in the reconstitution studies. The control and acetylated stripped chromatin had identical CD and thermal denaturation properties, as did their reconstitutes with rat thymus H1. Reconstitutes of the two chromatins with chicken erythrocyte H1 had similar CD properties, but thermal denaturation studies showed that the acetylated reconstitute was destabilized compared to the control reconstitute. Reconstitutes of both chromatins with chicken erythrocyte H1 had a more condensed and stabilized structure than the reconstitutes with rat thymus H1. Thus, acetylation caused a decrease in the stability of chromatin in the presence of erythrocyte H1, but more marked differences were detected in the structure of stripped chromatin after reassociation with different H1 preparations.     

Effect of polyethylene glycol on the thermal stability of green fluorescent protein

Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Some polymers, such as polyethylene glycol, are often used as modifiers of characteristics of biological macromolecules, to improve the biochemical activity and stability of proteins or drug bioavailability. The aim of this study was to evaluate the thermal stability of GFP in the presence of different PEG molar weights at several concentrations and exposed to constant temperatures, in a range of 70–95°C. Thermal stability was expressed in decimal reduction time. It was observed that the D‐values obtained were almost constant for temperatures of 85, 90, and 95°C, despite the PEG concentration or molar weight studied. Even though PEG can stabilize proteins, only at 75°C, PEG 600 and 4,000 g/mol stabilized GFP. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.     

HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases

HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases. Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis. Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings. The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.     

Studies on the conformational properties of the high-mobility-group chromosomal protein HMG 17 and its interaction with DNA

The conformation of the non-histone chromatin protein, HMG 17, has been studied using circular dichroism, infrared and nuclear magnetic resonance spectroscopies, and by small-angle scattering. The results show that in free solution this protein has little or no secondary or tertiary structure in contrast to the other high-mobility-group proteins, HMG 1 and 2, which exhibit highly ordered structures. Protein HMG 17 binds to calf thymus DNA in an ionic-dependent manner, precipitating the DNA at high protein/DNA ratio. The nuclear magnetic resonance data suggest that the principle DNA-binding segment of HMG 17 is that between about residues 15 and 40.     

Conformation of the HMG17-nucleosome complex

The nucleosome core binds more than two molecules of HMG17 at low ionic strength (8.9 mM Tris-HCl/8.9 mM boric acid/0.25 mM Na2EDTA, pH 8.3). Circular dichroism of the complexes showed only minor conformational changes of the nucleosome core DNA on binding of HMG17, with no detectable change in the histone secondary structure. The fluorescence of N-(3-pyrene) maleimide bound to -SH groups at Cys-110 of H3 histones in the core particle suggested that the structure of the histone octamer assembly changed little upon binding of HMG17 to the nucleosome. These observations support the idea that even a high level of HMG17 binding, e.g., four HMGs per nucleosome, alone, does not open up the core particle.     

Effect of proline to alanine mutation on the thermal stability of the all-beta-sheet protein tendamistat

The temperature dependent denaturation of wild-type tendamistat and of the proline-free triple mutant P7A/P9A/P50A was investigated using Fourier-transform infrared (FTIR) spectroscopy. Whereas the temperature-induced unfolding is reversible in the wild type, aggregation was observed for the proline-free tendamistat when studied under the same conditions. The midpoint unfolding temperature T(m) was found as 82.3+/-0.5 degrees C in (2)H2O. The thermal stability of the proline-free mutant is reduced by 15 degrees C as compared to the wild type. Changes in the strength of hydrogen bonding of tyrosine O-H groups upon unfolding and aggregation are reflected in small shifts of the C-C stretching mode of the aromatic ring near 1515 cm(-1). Evaluation of data from different infrared (IR) bands sensitive to changes in secondary structure as well as to changes in tertiary structure strongly supports a two-state model for the unfolding process of wild-type tendamistat.     

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.     

Increasing the thermal stability of an oligomeric protein, beta-glucuronidase.

The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.     

Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior.     

Human RAD52 protein has extreme thermal stability.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 associate into seven-membered rings. These rings can form higher order complexes. RAD52 binds to DNA breaks, and recent studies suggest that the higher order self-association of the rings promotes DNA end joining. Monomers of the RAD52(1--192) deletion mutant also associate into ring structures but do not form higher order complexes. The thermal stability of wild-type and mutant RAD52 was studied by differential scanning calorimetry. Three thermal transitions (labeled A, B, and C) were observed with melting temperatures of 38.8, 73.1, and 115.2 degrees C. The RAD52(1--192) mutant had only two thermal transitions at 47.6 and 100.9 degrees C (labeled B and C). Transitions were labeled such that transition C corresponds to complete unfolding of the protein. The effect of temperature and protein concentration on RAD52 self-association was analyzed by dynamic light scattering. From these data a four-state hypothetical model was developed to explain the thermal denaturation profile of wild-type RAD52. The three thermal transitions in this model were assigned as follows. Transition A was attributed to the disruption of higher order assemblies of RAD52 rings, transition B to the disruption of rings to individual subunits, and transition C to complete unfolding. The ring-shaped quaternary structure of RAD52 and the formation of higher ordered complexes of rings appear to contribute to the extreme stability of RAD52. Higher ordered complexes of rings are stable at physiological temperatures in vitro.     

Unwinding of DNA by nonhistone protein HMG1 and HMG2

The enzyme kinetic studies with endonucleases specific for single-stranded DNA and the thermal denaturation analyses of DNA showed that a high mobility group (HMG) nonhistone protein fraction HMG (1 + 2), composed of HMG1 and HMG2, has an activity to unwind DNA partially at low protein-to-DNA weight ratio. Isolated HMG1 and HMG2 have the same activity. Divalent cations such as Mg++ or Ca++ were necessary for the unwinding reaction. A peptide containing high glutamic and aspartic (HGA) region, isolated from the tryptic digest of HMG (1 + 2), unwound DNA depending on the presence of Mg++ or Ca++, suggesting that the HMA region in HMG protein is the active site for the DNA unwinding reaction. Poly-L-glutamic acid, employed as a model peptide of the HGA region, showed the activity. Finally, mechanisms of the DNA unwinding reaction by the HMG protein and possible role of the divalent cations are discussed.