Analysis of ligand-binding data by lambda invariance testing.

A method for analyzing data for one or two classes of homogeneous binding sites is presented. It uses moments of exponentially depressed data. From these the binding parameters are calculated as a function of the depression. The estimated binding constants are given by those values for which the parameters are extrema. The method needs no starting guesses for the parameters since it calculates them instead of fitting them by minimizing an objective function. The method is quick; it provides estimated binding constants with good precision and verifies the number of classes by a component incrementation test.

Robust estimation in pulse fluorometry. A study of the method of moments and least squares.

Most laboratories use least-squares iterative reconvolution (LSIR) as a routine method for estimating decay parameters in pulse fluorometric data. It is shown here, however, that LSIR is very sensitive to small amounts of error in the data whenever two decays become too close to one another, or whenever analyses of three decays are attempted. In such cases, inferior methods of estimating integrals, small zero point shifts, or small errors in the measured exciting light will result in failures of least squares, where the method of moments, with moment index displacement and lambda invariance testing, will succeed. The method of moments is therefore robust with respect to such errors while least squares is not.

The organization of oligonucleosomes in yeast.

We have developed a method of preparing yeast chromatin that facilitates the analysis of nucleoprotein organization. Yeast chromatin, isolated as an insoluble complex, is digested with micrococcal nuclease and fractionated into major insoluble and soluble fractions. No nucleosomal repeat is seen early in digestion for the insoluble fraction. Nucleosomal complexes of the soluble fraction are excised by nuclease in a distinctive non-random pattern; they are markedly depleted in mononucleosomes. When we analyze the soluble material by high resolution native electrophoresis, we find that the nucleoproteins resolve into two bands for each DNA multimer of the nucleosomal repeat. Our results suggest that there are structural similarities between bulk yeast chromatin and chromatin configurations found in transcribing genes of complex eukaryotes.

Immunochemical detection of chromosomal protein HMG-17 in chromatin subunits.

Chromosomal protein HMG-17, purified from calf thymus, has been used to elicit specific antibodies in rabbits. Specific serological reaction between the antigen and the antisera is demonstrated by solid-phase radioimmunoassay and by competitive inhibition assays. The antisera did not cross-react with histones or other chromosomal HMG proteins. The antisera bound specifically to chromatin subunits isolated from HeLa cells, demonstrating that it may be used to study the in situ organization of this chromosomal protein. Chromatin purified from HeLa nuclei was digested with micrococcal nuclease, and the resulting mono- and oligonucleosomes were fractionated on a sucrose gradient. Analyses of the content of chromosomal proteins HMG-1, HMG-17, and H4 in different size nucleosomal particles, by the solid-phase radioimmunoassay, reveal that the distribution of HMG-17 was the same as that of H4, but different from that of HMG-1.

High mobility group proteins of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains four proteins having amino acid compositions typical of the high mobility group (HMG) proteins. Three of these are eluted from chromatin by 0.35 M NaCl; one is not, but it is eluted by 0.25 N HCl. It follows that HMGs cannot, in general, be defined by extractability criteria. Gel mobilities and amino acid compositions indicate that yeast and animal HMGs have diverged markedly.

Analysis of nonexponential fluorescence decay data by a method of moments.

A method of moments is presented for the analysis of convoluted fluorescence decay data when the impulse response function is given by f(t) = alpha exp (-At - Bt1/2). Examples of this method are given using both simulated and measured fluorescence decays. It is also shown that this method, used with moment index displacements, will correct for light-scatter leakage, zero-point time shifts, and slow lamp drift.

Preparative gel electrophoresis: detection, excision, and elution of protein bands from unstained gels.

Methods are described for localizing proteins in unstained gels and accurately excising the regions containing them. A gel elutor, which is all glass with Lucite fittings, is also described. The elutor removes proteins from gels with a high yield and concentrates the protein in a small volume. The elutor is simple and very easy to use. A way is presented for avoiding the oxidation of methionine and cysteine during preparative electrophoresis.

Yeast inner histones and the evolutionary conservation of histone-histone interactions.

The inner histones of the yeast, Saccharomyces cerevisiae, have been isolated and identified by their amino acid compositions. H4 appears to be close to its calf and pea counterparts. H2a, H2b, and H3 have diverged. The isolation of the histones was accomplished by consecutive slab-gel fractionation, and a number of novel features of the method are described. These appear to be generally useful for preparing many types of protein. The binding pattern of the yeast inner histones is identical to the binding pattern for calf and for pea histones. Data on interspecies complexing indicate that the surfaces across which the histones interact are very highly conserved.

Immunological relatedness of high mobility group chromosomal proteins from calf thymus.

The non-histone proteins HMG-1, HMG-2, HMG-3, HMB-8, HMG-14, and HMG-17 (Goodwin, G. H., SANDERS, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14) were purified from calf thymus. The apparent molecular weights on polyacrylamide gels run in the presence of sodium dodecyl sulfate of the high mobility group (HMB) proteins were determined. Those for HBG-1 and HMG-2 agreed with the molecular weights determined by sedimentation; that for HMG-17 was anomalously high. Antibodies against HMG-1 were elicited in rabbits. The interaction between HMG-1 and anti-HBG-1 was measured by quantitative precipitation and by the microcomplement fixation technique. Quantitative microcomplement fixation assays revealed that the indices of dissimilarity between HMG-1 and HMG-2, HMG-3, HMG-8, HMG-14, and HMG-17 were 2.0, 1.0, 3.8, 10.0, and 6.1, respectively. These correspond to 6%, 0%, 12%, 20%, and 16% sequence difference between HMG-1 and the other five HMG proteins, although the immunological distance between HMG-1 and HMG-14 may be too large to allow a good correlation between the sequence and the immunological reaction. Antibodies to HMB-1 bind to chromatin purified from calf thymus. Therefore, we suggest that the in situ organization of HMG proteins in chromatin and chromosomes may be studied by serological techniques.

Cross-complexing pattern of plant histones.

Pea histones H2a, H2b, H3, and H4 have been isolated and their interactions studied by fluorescence anisotropy, light scatter, and circular dichroism. Histones H3 and H4 are almost identical in plants and animals, but plant histones H2a and H2b differ markedly from their mammalian counterparts. Pea H2b has a molecular weight approximately 20% greater than that of calf thymus H2b; the amino acid compositions of the two proteins are different. Calf thymus H2a exists as a single molecular weight species, while pea H2a exists as two species which differ by about 1500 daltons. The larger plant H2a is about 19% greater in molecular weight than calf thymus H2a. The smaller is about 8% greater. Despite these differences between calf and pea histones, the strong interactions between histone pairs H3 and H4, H2b and H4, and H2a and H2b, previously demonstrated for calf histones, also exist for pea histones. There are also weak interactions between pea H2a and H4 and between pea H2b and H3, and an interaction of intermediate strength between H2a and H3. The cross-complexing pattern of the plant histones is therefore the same as that reported for calf thymus histones [D'Anna, J. A., Jr., and Isenberg, I. (1974), Biochemistry 13, 4992], despite the dissimilarities of H2a and H2b.

Interactions between the subfractons of calf thymus H1 and nonhistone chromosomal proteins HMG1 and HMG2.

The nonhistone chromosomal proteins, HMG1 and HMG2, interact with the various subfractions of calf thymus H1 with a high degree of specificity. Subfractions 1b and 2 interact very strongly with HMG1 to form heterodimers. In contrast, subfractions 3a and 3b interact much more weakly. HMG2 interacts with 3a and 3b but shows no detectable complexing with 1a and 2.

Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.

Physical studies of the nonhistone chromosomal proteins HMG-U and HMG-2.

The nonhistone chromosomal proteins, HMG-1 and HMG-2, have a folded conformation, with a high alpha-helical content, over a wide pH range. At high and low pH values, the molecules unfold. Both molecules contain cysteine and tryptophan. The tryptophans appear to be buried in the folded form. HMG-1 shows aggregation at pH 5.7, as does HMG-2 at pH 9.0. The folded form is insensitive to high concentrations of salt, suggesting that charge-charge interaction plays no role in stabilizing the tertiary structure.

On the analysis of circular dichroic spectra of proteins.

A new method is presented for analyzing circular dichroism spectra. The method employs integrals over the data and calculates the alpha-helical, beta-sheet, and random coil content of the proteins from such integrals. It is shown that the analyzed alpha-helical content is usually reliable to within 5%, beta-sheet values are somewhat less reliable, and random coil values are least reliable. Curve fitting techniques are shown to be misleading. The method has a number of advantages over existing procedures.