Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS-protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14-200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS-protein complexes: (i) for proteins in the 14-65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS-protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.     

Analysis of the electrophoretic migration of DNA frayed wires

We analyzed the electrophoretic behaviour of the unusual multi-stranded DNA complexes, frayed wires, in polyacrylamide gels under non-denaturing conditions. Frayed wires arise from the association of several strands of a parent oligonucleotide that possesses long terminal runs of consecutive guanines. According to the structural model proposed for frayed wires, there are two distinct conformational domains, a guanine stem and single stranded arms displaced from the stem. The presence of the two domains affects the electrophoretic migration of the frayed wires, resulting in a greater retardation compared to that of double stranded DNA of the same molecular weight. The degree of retardation is determined by the relative length of the stem and the arms; the complexes with longer arms display a stronger dependence on the total molecular weight. Reptation plots (mobility x molecular weight vs. molecular weight) were used to study the electrophoretic behaviour of frayed wires that arise from the different parent oligonucleotides. The plots are unique for each type of frayed wire. The characteristic parameter, the position of the maximum of the reptation plot, depends on the type of the frayed wire as well as the total gel concentration. The plots become similar when we replot the mobility data taking into account only the single stranded arms of the frayed wires. The positions of the maximum and the overall shape are very close for the four types of frayed wires studied.     

Effect of sequence on the structure of three-arm DNA junctions

We have investigated the geometry of a number of three-arm branched DNA molecules by measuring the relative electrophoretic mobilities of analogues of each junction in which one pair of arms is extended. In general, the mobilities of three species of three-arm junctions in which the duplex arms are extended pairwise differ in the presence of Mg2+. This effect is eliminated by the absence of Mg2+ or by an increase in temperature, leading us to conclude that the three-arm DNA junctions are not 3-fold symmetric, because of either preferential stacking or asymmetric kinking of the arms at the branch that occurs in the presence of Mg2+. The geometry of the junction is governed by the base sequence at the branch and 1 bp removed from the branch. The pairwise elongated analogues of junctions that contain identical base pairs at the branch or 1 bp from the branch show mobility differences; when both positions have the same sequence no mobility differences are detected even in the presence of Mg2+. Formation of a branch in three-arm DNA junctions can be seen to produce a strain or deformation that propagates about one turn of the helix from the branch, leading thymines in this region to become hyperreactive to osmium tetraoxide. Surprisingly, the effect is independent of the presence or absence of metal cations. The structure of the three-arm junction is thus quite different in character from that of four-arm junctions both in the presence and absence of high concentrations of metal cations.     

Human dynein and sperm pathology

Human spermatozoa with normal structure and with different axonemal deficiencies (absence of axoneme, of arms, or of central structures) were studied by electron microscopy, SDS-polyacrylamide gel electrophoresis, and ATPase activity measurements. Normal human sperm possess a complement of high molecular weight polypeptides with an electrophoretic migration similar to that of sea urchin and other mammalian sperm dyneins. Human high molecular weight bands are numbered one to four in order of increasing of electrophoretic mobility; all of them are absent in spermatozoa that lack axoneme. The absence of doublet arms, coincides with the absence of bands 2, 3, and 4; the absence of central structures coincides with a reduction in intensity of band 2. In the latter two abnormal conditions, band 1 has an increased intensity. The data are tentatively interpreted by attributing the polypeptides forming bands 3 and 4 to the arm structure, whereas band 2 is supposed to contain a mixture of polypeptides localized in the arms and in the central structures; these abnormal sperm contain modified polypeptides which gather in band 1. Histochemical ATPase stainings indicate that this enzyme is localized mainly in the doublet arms and, to a minor extent, in the central structures.     

Analysis of branched nucleic acid structure using comparative gel electrophoresis

Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.     

An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5( 1.00) allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model.     

A model of random fiber networks that describes the sieving of particles during gel electrophoresis.

A model is presented to describe the sieving of particles during gel electrophoresis by considering the movement of a spherical particle through a random network of straight, rigid fibers. The movement of the particle through the network is approximated by a discrete model of the network composed of parallel planes containing fibers through which the particle must pass. Unlike previous models this model does not assume that the rate of movement is proportional to the proportion of cross-sectional area available to the particle. The results provide a new justification for approximately linear Ferguson plots and suggest that for large particles, Ferguson plots may become nonlinear.     

Heterogeneity of enterotoxin-like protein extracted from spores fo Clostridium perfringens type A.

Enterotoxin-like protein was extracted from spores of three enterotoxin-positive and three enterotoxin-negative strains of Clostridium perfringens type A by urea/mercaptoethanol, alkaline mercaptoethanol and alkaline dithiothreitol. Disc immunoelectrophoresis demonstrated that three distinct enterotoxin-like proteins could be extracted. In 7% acrylamide gels, type I, type II, and type III enterotoxinlike proteins had relative mobilities of 0.52, 0.63, and 0.73 respectively. In contrast to disc immunoelectrophoresis, immunoelectrophoresis in agar gel demonstrated identical electrophoretic properties for the various entertoxin-like proteins. Immunoelectrofocusing experiments gave isoelectric points of 4.43, 4.43, 4.36, and 4.52 for purified entertoxin and type I, type II, and type III enterotoxin-like proteins respectively. Ferguson plots (i.e., log relative mobility versus acrylamide concentration) yielded nonparallel lines which intersected at a nonsieving concentration of acrylamide indicating that the various species of enterotoxin-like protein differed in size. Estimation of the molecular weight of purified enterotoxin and the three species of enterotoxin-like protein was done by comparing the slopes obtained in Ferguson plots with those obtained using proteins of a known molecular weight. Molecular weights of 38000, 36500, 23000, and 15400 were obtained for purified enterotoxin, type I, type II, and type III enterotoxin-like protein respectively. Collectively, the evidence indicates that fractionation of the different species of enterotoxin-like protein was due primarily to differences in their size, and that different forms of enterotoxin-like protein can be extracted from spores of different strains of C. perfringens type A.     

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat. General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca2+ (in the absence of presence of calmodulin) or independent of these factors. In the present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

Intrinsic protein phosphorylation in synaptic plasma membrane fragments from the rat General characteristics and migration behaviour on polyacrylamide gels of the main phosphate acceptors

Preparations enriched in synaptic membrane fragments from rat cerebral cortex contain protein kinases which phosphorylate membrane proteins in reactions dependent on cAMP, Ca²⁺ (in the absence or presence of calmodulin) or independent of these factors. In these present work characteristics of the main phosphorylated acceptors were studied and compared with the results of other investigations. Apparent molecular weights were estimated by determining electrophoretic mobility on gels of different acrylamide concentration. Irregular migration behaviour was detected by measuring free mobilities from Ferguson plots. Certain phosphate acceptors were found to exhibit anomalously low free mobilities and it was concluded that estimates of molecular weight for these acceptors were unreliable.     

Assembly and characterization of five-arm and six-arm DNA branched junctions.

DNA branched junctions have been constructed that contain either five arms or six arms surrounding a branch point. These junctions are not as stable as junctions containing three or four arms; unlike the smaller junctions, they cannot be shown to migrate as a single band on native gels when each of their arms contains eight nucleotide pairs. However, they can be stabilized if their arms contain 16 nucleotide pairs. Ferguson analysis of these junctions in combination with three-arm and four-arm junctions indicates a linear increase in friction constant as the number of arms increases, with the four-arm junction migrating anomalously. The five-arm junction does not appear to have any unusual stacking structure, and all strands show similar responses to hydroxyl radical autofootprinting analysis. By contrast, one strand of the six-arm junction shows virtually no protection from hydroxyl radicals, suggesting that it is the helical strand of a preferred stacking domain. Both junctions are susceptible to digestion by T4 endonuclease VII, which resolves Holliday junctions. However, the putative helical strand of the six-arm junction shows markedly reduced cleavage, supporting the notion that its structure is largely found in a helical conformation. Branched DNA molecules can be assembled into structures whose helix axes form multiply connected objects and networks. The ability to construct five-arm and six-arm junctions vastly increases the number of structures and networks that can be built from branched DNA components. Icosahedral deltahedra and 11 networks with 432 symmetry, constructed from Platonic and Archimedean solids, are among the structures whose construction is feasible, now that these junctions can be made.     

Computer-aided analysis of DNA curves on transverse gradient gels.

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Brownian diffusion and electrophoretic transport of double‐stranded DNA in agarose gels

The field free diffusion constant and the electric field dependence of the electrophoretic mobility and molecular orientation of DNA samples from 5 to 164 kilobase pairs in agarose gels from 0.5 to 2% have been measured by fluorescence recovery after photobleaching and birefringence. In conditions where the reptation predictions hold for the field free diffusion, they partially fail for the DNA size dependence of the low field limit of the electrophoretic mobility. The linear field dependencies of the electrophoretic mobility and orientation factor seem to favor the biased reptation model with fluctuations over the standard biased reptation model, which predicts a quadratic field dependence. The quantitative analysis of the molecular parameters shows, however, that most experiments have been carried out at values of the field where the difference between the two models may be less conclusive. The pore size dependence of the different quantities has been given a particular attention and the role of the distribution of pore sizes in the departures from the reptation predictions is discussed. © 1999 John Wiley & Sons, Inc. Biopoly 50: 45–59, 1999     

Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.     

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.     

Hydrophobic and ionic effects upon the electrophoretic mobilities of the subunits of coupling factor 1 from mitochondria

A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was used to investigate certain properties of the subunits of the beef heart mitochondrial ATPase, (native F1, nF1). By examining the affects of urea concentration and acrylamide concentration upon the electrophoretic mobilities of the polypeptides comprising the nF1 enzyme, we have obtained conditions under which all five subunits are simultaneously resolved when the discontinuous buffer system of Laemmli is used (U. K. Laemmli (1970) Nature (London) 277, 680-685). The determination of the apparent molecular weights by analysis of Ferguson plots (K. A. Ferguson (1964) Metabolism 13, 985-1002) revealed that the addition of urea to the SDS gels resulted in a decrease in the apparent molecular weight of the beta subunit. A dramatic increase in the apparent molecular weight of the delta subunit was also brought about by the presence of urea in the SDS gels. In addition, the apparent molecular weight of both the alpha and the beta subunits was dependent upon the acrylamide concentration used, indicating that these subunits contain either areas highly resistant to denaturation by the combined action of urea and SDS, or covalent modifications leading to anomalous electrophoretic mobility. The results of experiments in which urea analogs were used indicate that the interactions of urea with the beta subunit involve the formation of hydrogen bonds between urea and regions of this subunit. On the other hand, the interactions of urea with the delta subunit are primarily of a hydrophobic nature, suggesting that these interactions could involve domains of the delta subunit required for binding of the coupling factor to the mitochondrial membrane.     

Asymmetric structure of a three-arm DNA junction

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous degradation of rat liver chromatin studied by agar gel electrophoresis of nuclei

Direct agar gel electrophoresis of incubated rat liver nuclei revealed that most of the chromatin is rapidly converted to stable, large fragments, showing identical electrophoretic mobility. Short and long term incubation gave the same results. The analysis of deproteinized DNA under nondenaturing as well as denaturing conditions showed, however, a correlation between the DNA size pattern and the time of incubation. Our data on the persistance of large and uniform in size chromatin fragments despite the presence of cleaved DNA in them may indicate naturally footprinted regions of chromatin, implying most probably some strong ordered interaction of chromatin constituents. It seems that some substantial unknown features of higher order structure of chromatin are preserved in rat liver nuclei isolated and digested under the experimental conditions used.     

Comparative structural analyses of corticosteroid binding globulin

Some physicochemical characteristics of corticosteroid binding globulin (CBG) in several species have been determined. Molecular radii were determined from Ferguson plots and were used in conjunction with sedimentation coefficients determined by sucrose density gradient centrifugation to calculate the molecular weights of the CBG. These were found to range from 44,200 (dog) to 60,000 (turtle) for most species. The squirrel monkey was found to have a molecular weight twice that of other species (119,800). Purified CBG was prepared from human, rat, and guinea pig sera. The molecular weights of the purified material, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate, were in excellent agreement with those determined by Ferguson analysis. Careful examination of the purified proteins by electrophoresis at pH 8.3 revealed that each consisted of two closely related electrophoretic variants. Tryptic peptides were prepared from the purified proteins and separated by reversed phase HPLC chromatography. The peptide patterns were identical for the three proteins with the exception of three hydrophilic peptides. Amino terminal sequence analysis of the rat and human proteins revealed no apparent homology, however. The immunologic relatedness of the three purified proteins was also examined, but no crossreactivity was observed. The results obtained suggest that while the molecular size and hydrophobicity of peptides have been conserved across species considerable surface differences must exist.