Gonadotropin and subunit conformation.

Circular dichroic spectra have been obtained and resolved for the gonadotropins, ovine pituitary luteinizing hormone and human chorionic (urinary) gonadotropin, their subunits and glycopeptides. Much of the gonadotropin ellipticity above 250 nm can be attributed to the disulfide chromophore, although there are discernible contributions from tyrosyl and phenylalanyl residues as well. Of the two dissimilar subunits, the β-subunit makes the greatest contribution to the near-ultraviolet circular dichroic spectrum of the gonadotropins. From the position of the 0-0 tyrosyl band, i.e., 286–287 nm, one can ascertain that at least some of the tyrosyl residues of the gonadotropins are located in a hydrophobic environment. A positive circular dichroic extremum at 232.5 nm, present in luteinizing hormone but not in chorionic gonadotropin, can be ascribed to the α-subunit and probably results from tyrosines 21 and/or 30 in luteinizing hormone.An analysis of the circular dichroic difference spectrum above 230 nm, generated by subtracting the sum of the molecular ellipticities of the respective subunits from the molecular ellipticities of each gonadotropin, indicates that the local environment of disulfides and of tyrosyl residues is altered when gonadotropins dissociate. Circular dichroic difference spectra between the two α-subunits and between the two β-subunits indicated major contributions from- tyrosyl residues, presumably arising from tyrosyl substitutions.Between 200 and 230 nm, both gonadotropins exhibit negative circular dichroic extrema. The extremum occurs at 210 nm for luteinizing hormone and at 207.5 nm for chorionic gonadotropin. Each extremum can be described by two negative resolved bands, one at 215 nm and the other between 207 and 208.5 nm. The 215-nm resolved band is assigned to the peptide chromophore in a β-pleated sheet conformation and there is no evidence of α-helicity. The lower-wavelength resolved band is believed to have a significant contribution from the N-acetyl groups of glucosamine, galactosamine, and sialic acid, particularly since the glycopeptide fractions, prepared from each gonadotropin by digestion of the S-carboxymethyl derivatives with Pronase, exhibited a negative circular dichroic extremum at about 207 nm.The extent of β-structure in both gonadotropins is estimated to be about 28% whereas the separated subunits contain less β-structure, e.g., about 21 and 13% for the α- and β-subunits, respectively. The sum of the subunit β-structure, corrected for the respective molecular weight of each subunit, is about 17%. This is substantially less than that of the native hormone, thus indicating that significant conformational changes occur during gonadotropin dissociation to the biologically inactive subunits. Also, part of the gonadotropin β-structure may arise from intermolecular hydrogen bonding involving a pleated sheet arrangement between the subunits.     

CD of ethidium bromide complexes with normal and electrophoretically anomalous DNA restriction fragments

The equilibrium binding of ethidium bromide (EB) to two small 147 base-pair (bp) DNA restriction fragments, which exhibit different mobilities in polyacrylamide gels, was investigated by CD. Two larger DNA restriction fragments and calf thymus DNA were also studied for comparison. Difference spectra were calculated by subtracting the spectrum of the pure DNA from the spectra of its DNA–EB complexes. The D/P ratios ranged from 0.03 to 1.0. The difference CD spectra of all fragments are characterized by bands with maxima near 310, 275, and 207 nm, and minima near 290, 253, 225, and 190 nm. The band near 310 nm, which has a shoulder at about 335 nm, has zero intensity at D/P ≤ 0.05, and rises to a plateau value, different for each fragment, at D/P ? 0.3 for large fragments (≥ 1400 bp), and D/P ～ 0.7 for the two small 147 bp fragments. The minimum near 290 nm is markedly blue shifted with increasing D/P, the wavelength of the extremum corresponding approximately to the wavelength of the uv absorption maximum of the DNA–EB complex. The negative amplitude of this band at D/P = 1.0 depends on the molecular weight of the DNA. The difference CD maximum near 275 nm is positive at low D/P ratios, increases and goes through a maximum at D/P = 0.06–0.1, and then becomes increasingly negative with increasing D/P. The amplitude of the negative ellipticity per added dye is constant at high D/P ratios, suggesting that the transition can be attributed to outside-bound EB molecules. The ellipticities at 310, 290, and 253 nm increase in absolute magnitude with increasing D/P at approximately the same rate, suggesting that all three bands are associated with the same optical and/or conformational transition. For the two small 147 bp fragments the fractional increases in amplitude of these bands parallel the fractional increase in length of the DNA upon binding EB, determined by electric birefringence measurements. The titration of the restriction fragments with EB was also followed by optical absorption. Two end points are observed, the first at a D/P ratio of ～ 0.1, reflecting the transition between intercalated and outside-bound dye molecules, and the second at D/P ? 1.0, the equivalence point of the titration.     

D-glucose binding increases secondary structure of human erythrocyte monosaccharide transport protein

Purified hexose transport protein ("band 4.5") from human erythrocytes, reconstituted in vesicles of its endogenous lipids, displays minima in its circular dichroism (CD) spectrum at 222 and 207 nm, a pattern diagnostic for alpha-helical content of proteins. Upon addition of D-glucose, a saturable increment of +10-12% in negative ellipticity at 222 nm is observed stereospecifically and reproducibly. Addition of L-glucose had no effect on the CD spectrum of the transport protein. Addition of cytochalasin B (CB), a reversible inhibitor of hexose transport, had no effect itself on transporter CD spectra, but restored the spectrum at 222 nm to its original value when added in the presence of D-glucose. The observed D-glucose-induced increase in ordered secondary structure is proposed to result from incorporation into the membrane of a segment of the transport protein originally at a membrane-water interface.     

Circular dichroism studies of an elastin crosslinked peptide.

Circular dichroic studies of a desmosine crosslinked peptide reveal a hitherto undescribed elastin spectrum possessing a weak negative band at 230–235 nm, a weak positive band at 215 nm, and a maximum negative band at 190 nm. The spectrum is sensitive to both pH and temperature displaying increased ellipticity of the 215-nm band at acidic pH and low temperature. The general shape of the spectrum and its behaviour toward temperature changes suggest the presence of an extended helical conformation. Susceptibility of insoluble elastin to digestion with chymotrypsin is increased tenfold at low temperature (4°C), supporting the contention that the conformational change of the type described above occurs in insoluble elastin. Such changes in conformation would result in increased availability of aromatic amino-acid resiudues to peptide bond cleavage.     

岩豆凝集素分子修饰与圆二色性的关系研究

天然态岩豆凝集素（ＭＤＬ）远紫外圆二色性（ＣＤ）谱显示２１６ｎｍ处单一负峰,是一种高β－折叠构象凝集素;近紫外ＣＤ谱呈现２８２ｎｍ处负峰和２６０～２７５ｎｍ及２９５ｎｍ处的负肩,经Ｎ－乙基顺丁烯二酰亚胺（ＮＥＭＩ）和对氯汞苯甲酸（ＰＣＭＢ）修饰ＭＤＬ的巯基,其近紫外ＣＤ谱未发生变化,远紫外ＣＤ谱仅发生细微变化,ＭＤＬ凝集活性保持不变;ＰＣＭＢ过量时,ＣＤ谱呈现典型的无规卷曲谱形,ＭＤＬ完全丧失凝集活性,去除ＰＣＭＢ后,活性又全部恢复．二硫苏糖醇（ＤＤＴ）修饰ＭＤＬ的二硫键并用碘乙酸（ＩＣＨ２ＣＯＯＨ）保护巯基,ＭＤＬ远紫外ＣＤ谱２１６ｎｍ处的负峰红移至２２５ｎｍ,且显著减小;同时,近紫外ＣＤ谱２８２ｎｍ处负峰几乎消失,两负肩分别保持完整,分子中α－螺旋降低,无规卷曲增加较多,ＭＤＬ凝集活性未发生变化．用Ｎ－溴代丁二酰亚胺（ＮＢＳ）修饰ＭＤＬ分子中的色氨酸,导致２１６ｎｍ负峰蓝移至２０８ｎｍ且变小,分子中无规卷曲和α－螺旋增加,β折叠减少,近紫外ＣＤ谱２９５ｎｍ负肩消失,２８２ｎｍ负峰红移至２８７ｎｍ,ＭＤＬ凝集活性完全丧失．     

Circular dichroism of cytochrome oxidase, cytochrome b566, and cytochrome c in beef heart mitochondrial membrane fragments

1. Circular dichroism spectra of the cytochromes in membrane fragments derived from sonicated beef heart mitochondria have been obtained in the wavelength region 400–480 nm in which the major absorbance maxima of the heme prosthetic groups are found.

2. 2. Cytochrome oxidase in the mitochondrial membrane fragments has a band of positive ellipticity at 426 nm in the oxidized form and a pronounced band of positive ellipticity at 445 nm in the reduced form. The reduced-minus-oxidized difference molar ellipticity at 445 nm, Δ[θ]₄₄₅ is 3.0·10⁵ degree·cm⁻²·dmole⁻¹ heme a for membrane-bound oxidase compared to 1.6·10⁵ degree·cm⁻²·dmole⁻¹ heme a for the purified oxidase. The membrane-bound oxidase in the reduced form also appears to have a band of negative ellipticity at 426 nm not found in the purified oxidase.

3. 3. When reduced with succinate in the presence of cyanide and oxygen, cytochrome oxidase in the membrane fragments has a positive band at 442 nm very similar to that observed with the purified oxidase.

4. 4. Cytochrome c, which has a positive band at 426 nm in the purified form when reduced, appears to have a negative band at this wavelength in the mito-chondrial membrane fragments which contributes to the pronounced negative band at 426 nm observed in the membrane fragments reduced with succinate in anaerobiosis. There is no evidence for a contribution to the CD spectra of the membrane fragments from cytochrome c₁ or from cytochrome b₅₆₁ in either the oxidized or the reduced form.

5. 5. Cytochrome b₅₆₆ in the mitochondrial membrane fragments has no detectable CD spectrum in the oxidized form, but has a small positive band at 427 nm and a small negative band at 436 nm in the reduced form. The same CD spectrum is observed with cytochrome b₅₆₆ reduced with succinate in the presence of antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide. The same increase in positive ellipticity is observed at 427 nm in the mitochondrial membrane fragments, treated with oligomycin to restore energy coupling, when cytochrome b₅₆₆ is reduced with succinate in the energized membrane, as is observed in the inhibitor-treated membrane fragments. The absence of a pronounced conformational change in cytochrome b₅₆₆ on energization, as revealed by its CD spectrum, favors the concept that its reduction by succinate in the energized state is due to reversed electron transport rather than an intrinsic shift in the cytochrome's midpoint redox potential.

Tyrosine optical activity as a probe of the conformation and interactions of fibronectin

A very intense negative band is observed at ～ 183 nm in the CD spectrum of fibronectin from bovine plasma. This transition has not previously been reported, probably because it occurs in a spectral region that has not been readily accessible in earlier studies. At longer wavelength, the observed CD is very similar to spectra reported for human and chick material, having positive bands at ～230 and ～200 nm, and a negative band at ～215nm. The low molar ellipticity of the negative band ([θ] ≈ ?2.5 × 10³ deg cm² dmol^?1) suggests little α-helix or β-sheet structure. The new transition, and the two positive bands at higher wavelength, do not correspond to known transitions of the peptide backbone, but all three are present in the CD of N-acetyltyrosineamide. It is therefore suggested that the observed CD behavior of fibronectin arises predominantly from the optical activity of tyrosine side chains. The contribution of this side-chain optical activity to the CD of other proteins is discussed. On raising pH to ionize tyrosine residues, the positive CD band at ～230 nm is lost in both N-acetyltyrosineamide and in fibronectin. The spectral change is fully reversible in the model compound, but only partially reversible in fibronectin. From this evidence, and the magnitude of the 183-nm band, it is suggested that some or all of the tyrosine residues in fibronectin may be present within ordered domains. The possible role of S? S bonds in maintaining tertiary structure is discussed. The interaction of fibronectin with heparin is accompanied by a large increase in the 183-nm band and by slight enhancement of the negative band at 215 nm, consistent with some limited formation of β-sheet. Present results indicate that CD may be of considerable value in characterization of the molecular organization and biologically relevant interactions of fibronectins and of related glycoproteins of the extracellular matrix.     

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.     

Conformational changes of type II regulatory subunit of cAMP-dependent protein kinase on cAMP binding

The effect of cAMP on the conformation of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from bovine heart was investigated by UV-difference and circular dichroism (CD) spectroscopy. The UV-difference spectrum of RII with and without cAMP showed a positive band around 278 nm and a negative band at 256 nm. Similarly, cAMP enhanced the ellipticity of RII in the region between 280 and 300 nm and decreased that between 250 and 280 nm. In addition, cAMP transformed the far-UV CD spectrum of RII from that of a negative band minimally at 209 nm with a shoulder at 223 nm to one with two minima at 222 and 211 nm. These data show that cAMP induces conformational changes of RII upon binding. Such structural changes may be the basis of activation of cAMP-dependent protein kinases by cAMP.     

Temperature dependence of the optical activity of human serum low density lipoprotein. The role of lipids.

Low density lipoprotein (LDL) (1.024-1.045 G/cm3) was prepared by ultracentrifugal flotation from serum of normal fasting subjects. Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra in the ultraviolet region were measured at 2, 25, and 37 degrees on LDL, lipid extracted from LDL, and on pure component lipids. All exhibit reversible, temperature-dependent optical activities. Sphingomyelin has a strong negative CD band around 195 nm. Cholesterol and cholesteryl esters have a CD minimum at 208 nm. They have positive CD bands around 201 and 198 nm which decrease sharply and become negative at 198 and 193 nm, respectively. The CD of the total lipid extract of LDL is negative and drops monotonically below 200 nm. Thus, the lipid moiety could account for the increasing negativity of the CD of LDL below 195 nm. After subtraction of the ellipticity corresponding to amounts of lipids in organic solvents equivalent to those found in LDL, the 208-210 nm trough of LDL diminishes markedly. This is accompanied by a blue-shift of the extrema from 195-196 to 193 nm and an increase in the magnitude of the positive ellipticity. The fractions of helix and of beta form in the protein, determined by the method of Y. H. Chen, J. T. Yang, and K. H. Chau ((1974), Biochemistry 13, 3350), in the wavelength interval of 250-240 nm, remain essentially unchanged between 2 and 37 degrees. These observations suggest that a substantial part of the thermal change in the CD spectrum of LDL between 208 and 210 nm may be attributable to lipids.     

Near-UV circular dichroism of trypsin inhibitor of adzuki beans attributable to disulfide groups.

The trypsin inhibitor of adzuki (Phaseolus angularis) beans shows a CD spectrum with a negative extremum at 280 nm and a positive shoulder around 245 nm. Since the inhibitor lacks tryptophan and tyrosine, the observed CD spectrum can be attributed to the six disulfide groups in the molecule. The CD features completely disappeared on reduction of the disulfide groups, and converged into a single negative extremum at 270 nm when the groups were modified to form mixed disulfides with glutathione. These observations of the CD properties of the inhibitor strongly suggest the presence of disulfide groups constrained with respect to their dihedral angles.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Differential scanning calorimetric studies of aqueous dispersions of phosphatidylcholines containing two polyenoic chains

The structure of the lectin discoidin I has been studied by circular dichroism and fluorescence spectroscopy. A positive ellipticity band at 224 nm is detected in the CD spectrum of discoidin I. The fluorescence spectra show a defined shoulder at 325 nm that through acrylamide quenching has been associated with a displaced tryptophan residue partly buried in the discoidin I molecule. This tryptophan could also be responsible for the 224 nm positive band of the CD spectrum. These spectroscopic characteristics of discoidin I indicate the existence of structural homologies with fibronectin, where the optical activity of aromatic chromophores has been associated with the positive ellipticity band at 227 nm. The CD adjust parameters and theoretical secondary structure predictions show that discoidin I is a molecule with a low content of alpha-helix and beta-strand and high content of beta-turn structures, similar to other lectins.     

Spectroscopic studies on the structural organization of the lectin discoidin I: homologies with fibronectin

The structure of the lectin discoidin I has been studied by circular dichroism and fluorescence spectroscopy. A positive ellipticity band at 224 nm is detected in the CD spectrum of discoidin I. The fluorescence spectra show a defined shoulder at 325 nm that through acrylamide quenching has been associated with a displaced tryptophan residue partly buried in the discoidin I molecule. This tryptophan could also be responsible for the 224 nm positive band of the CD spectrum. These spectroscopic characteristics of discoidin I indicate the existence of structural homologies with fibronectin, where the optical activity of aromatic chromophores has been associated with the positive ellipticity band at 227 nm. The CD adjust parameters and theoretical secondary structure predictions show that discoidin I is a molecule with a low content of α-helix and β-strand and high content of β-turn structures, similar to other lectins.     

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.     

Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145 tyrosine or beta 37 tryptophan

The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form. It has been suggested that the negative CD band is due to the environmental alteration of Tyr-alpha 42 or Trp-beta 37 at the alpha(1)beta(2) contact upon deoxygenation. To evaluate the contributions of the aromatic residues at the alpha(1)beta(2) contact and the penultimate tyrosine residues of the alpha and beta subunits (alpha 140 and beta 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-alpha 42, rHb His-beta 37, and rHb Thr-beta 145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-alpha 140-->His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T-->R transition (Hill's n > 2.0). The contributions of Tyr-alpha 42, Trp-beta 37, Tyr-alpha 140, and Tyr-beta 145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively. These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R-->T transition is primarily responsible for the negative CD band.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., K?gi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.     

Aspects of secondary and quaternary structure of surfactant protein A from canine lung

The results of a large number of studies indicate that pulmonary surfactant contains a unique protein whose principal isoform has a molecular weight of about 30,000, and whose presence in surfactant is associated with important metabolic and physicochemical properties. This protein, SP-A, as isolated from canine surfactant, contains a domain of 24 repeating triplets of Gly-X-Y, similar to that found in collagens. These studies were undertaken to determine whether SP-A forms a collagen-like triple helix when in solution, and to describe certain aspects of its size and shape. Our experiments were done on SP-A extracted by two different methods from canine surfactant, and on SP-A produced by molecular cloning. The results from all three preparations were similar. The circular dichroism of the complete protein was characterized by a relatively large negative ellipticity at 205 nm, with a negative shoulder ranging from 215 to 230 nm. There was no positive ellipticity, and the spectrum was not characteristic of collagen. Trypsin hydrolysis resulted in a fragment with peak negative ellipticity at about 200 nm, without the negative shoulder. Further hydrolysis of this fragment with pepsin resulted in a CD spectrum similar to that of collagen. The spectrum of the collagen-like fragment was reversibly sensitive to heating to 50 degrees C, and was irreversibly lost after treatment with bacterial collagenase. SP-A migrated on molecular sieving gels with an equivalent Stokes radius of 110 to 120 A, and had a sedimentation coefficient of 14 S. Using these data we calculate a molecular weight of about 700,000. The hydrodynamic characteristics can be approximated as a prolate ellipsoid of revolution having an axial ratio of about 20. We conclude that SP-A aggregates into a complex of 18 monomers, which may form six triple-helices. The shape of the complex is considerably more globular than collagen and is not consistent with end-to-end binding of the helices to form fibrous structures.     

A contribution of nonhistone proteins to the conformation of chromatin.

1. Changes in circular dichroism (CD) spectra and thermal melting profiles of guinea pigliver DNA reassociated with histones and/or nonhistone proteins from the cerebral of liver chromatin are described. 2. In the DNA-histone complex, positive ellipiticity in the CD spectrum at 260-300 nm is progressively lod by a red-shift of the crossover point at around 260 nm. DNA in this complex is thermally stabilised to a considerable extent, but not to such a full extent as is shown with DNA in native chromatin. 3. DNA-nonhistone complex in 0.14 M NaCl is, in contrast to DNA-histone complex, not precipitable by centrifugation at 20 000 X g. DNA in this complex shows only a slight reduction in ellipticity at 260-300 nm, and a very weak thermal stabilisation. 4. Characteristics in the CD spectrum of the native chromatin are most satisfactorily reproduced in the DNA-histone-nonhistone complex. These include a large decrease in ellipticity at 260-300 nm, a red-shift of the crossover point at around 260 nm, and a slight negative band at around 305 nm. Also, DNA in this complex is thermally stabilised to the extent comparable with DNA in the native chromatin. 5. Addition of nonhistone proteins to the preformed DNA-histone complex in 3 M urea renders a half of the complex, named DNA-histone(-nonhistone), unprecipitable upon centrifugation at 20 000 X g in 0.14 M NaCl. CD spectrum and thermal melting profile of the precipitable DNA-histone(-nonhistone) complex are similar to those of the DNA-histone-nonhistone complex, while in the unprecipitable DNA-histone(-nonhistone) comples, the ellipticity at 260-300 nm is significantly elevated and the highest melting transition (at 80 degrees C) is lacking. 6. The CD spectrum of native cerebral chromatin closely resembles that of unprecipitable DNA-histone(-nonhistone) complex, while in liver chromatin, the spec.trum is an intermediate between those of the unprecipitable and pn of chromatin by nonhistone proteins. Cerebral nonhistone proteins bind to DNA and to the DNA-histone complex more extensively than liver nonhistone proteins. 7. It is concluded that, although the basic conformation of DNA in native chromatin is determined largely by histones, nonhistone proteins also play an individual role. There is also an indication that nonhistone proteins exert an organ-specific modification of chromatin superstructure.