Comparative spin-label study of the dynamic structure of human hemoglobins A and F and the influence of the antibiotic, chloramphenicol

Hemoglobin A (HbA) and hemoglobin F (HbF) dynamic structures have been studied using spin-label ESR spectra analysis technique, which permits quantitative separation of slow macromolecular rotation (described by rotational correlation time, tau c) and fast anisotropic nitroxide radical motion (described by the 'order parameter', S). The hardly restricted motion of the maleimide spin-label reflects the overall macromolecular rotation and small dynamic structure differences between HbA and HbF were observed (tau c is equal to 26 and 27 ns, respectively). On the other hand, the dynamic equilibrium of the iodoacetamide spin-label demonstrates significant differences between beta- and gamma-chain C-terminus flexibility. Thus, there are different states of alpha,beta and alpha,gamma intersubunit contacts which may be expected to determine the different O2 affinity of HbA and HbF. The antibiotic, chloramphenicol, strongly affects the O2 affinity and the Hill constant of HbF, and also provides detectable changes of gamma-subunit C-terminus flexibility (tau c changes from 20 ns to 27 ns after chloramphenicol treatment of HbF), while the HbA tetramer structure remains almost unaffected. The HbF domain structure rearrangements are accompanied by a decrease of the steric restriction of the spin-label motion (S changes from 0.75 to 0.72).     

Lipid mobility and order in bovine rod outer segment disk membranes. A spin-label study of lipid-protein interactions

Lipid-protein interactions in bovine rod outer segment disk membranes have been studied by using a series of eight stearic acid spin-label probes which were labeled at different carbon atom positions in the chain. In randomly oriented membrane dispersions, the electron spin resonance (ESR) spectra of the C-8, C-9, C-10, C-11, C-12, C-13, and C-14 atom positional isomers all apparently consist of two components. One of the components corresponds closely to the spectra obtained from dispersions of the extracted membrane lipids, and the other, which is characterized by a considerably greater degree of motional restriction of the lipid chains, is induced by the presence of the protein. Digital subtraction has been used to separate the two components. The proportion of the motionally restricted lipid component is approximately constant, independent of the position of the spin-label group, and corresponds to 30-40% of the total spin-label spectral intensity. The hyperfine splitting of the outer maxima in the difference spectra of the motionally restricted component decreases, and concomitantly, the line widths increase with increasing temperature but change relatively little with increasing distance of the spin-label group from the polar head-group region. This indicates that the corresponding chain motions of the protein-interacting lipids lie in the slow-motion regime of spin-label ESR spectroscopy (tau R approximately 10(-8) S) and that the mobility of these lipids increases with increasing temperature but does not vary greatly along the length of the chain. The data from the hyperfine splittings also suggest the existence of a polarity gradient immediately adjacent to the protein surface, as observed in the fluid lipid regions of the membrane. The more fluid lipid component is only slightly perturbed relative to the lipids alone (for label positions 5-14, inclusive), indicating the presence of chain motions on the nanosecond time scale, and the spectra also reveal a similar polarity profile in both lipid and membrane environments. ESR spectra have also been obtained as a function of magnetic field orientation with oriented membrane samples. For the C-14 atom positional isomer, the motionally restricted component is observed to have a large hyperfine splitting, with the magnetic field oriented both parallel and perpendicular to the membrane normal. This indicates that the motionally restricted lipid chains have a broad distribution of orientations at this label position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural studies of human IgA1 and IgA2 immunoglobulins labeled with two different spin labels

The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure.     

Molecular dynamics in protein-single stranded DNA complexes. Two distinct nucleoside mobilities, in poly(deoxythymidylic acid)-poly-L-lysine complexes

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time tau -R = 2 ns, and the other one by a tau -R greater than or equal to 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Structure and flexibility of plasma fibronectin in solution: electron spin resonance spin-label, circular dichroism, and sedimentation studies

Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.     

Poly(adenylic acids) containing the antibiotic tubercidin -- base pairing and hydrolysis by nuclease S1.

Poly(adenylic acids) containing the antibiotic tubercidin (7-deazaadenosine) form double strands with poly(uridylic acid) by Watson-Crick base pairing. The stability of these complexes is enhanced by an increasing adenosine content of the polymers. Whereas poly(tubercidylic acid) can bind only one poly(U) chain, the copolymers of adenylic and tubercidylic acid bind a second strand of poly(U). The melting temperatures imply a triple strand formation in a similar geometry as found for poly(A).2poly(U). The diminished hypochromicity of those complexes suggests semi-Hoogsteen base pairs, caused by the lack of N-7 in the antibiotic. As found for poly(A).poly(U), the double-stranded poly(Tu).poly(U) is not hydrolyzed by nuclease S1. In contrast to the four regular homopolyribonucleotides the single-stranded poly(Tu) is cleaved very rapidly. This may be due to a great flexibility of the polynucleotide chain. Moreover TuMP does not inhibit the enzymic digestion. Both phenomena imply a mechanism for the antibiotic action of tubercidin on the polymer level.     

Interaction of metal ions with polynucleotides and related compounds. X. Studies on the reaction of silver (I) with the nucleosides and polynucleotides, and the effect of silver(I) on the zinc(II) degradation of polynucleotides

Potentiometric titration curves of the silver(I) complexes of cytidine, adenosine, and uridine show little uptake of base below pH 7, unlike the curve for silver(I)-guanosine, which shows extensive base uptake at neutral pH. This observation is correlated with spectrophotometric data showing little difference between the silver complex spectra of adenosine, cytidine, and uridine and the spectra of uncomplexed nucleosides, except at high pH, but showing a great difference between the silver complex spectra of guanosine and inosine and the corresponding uncomplexed nucleosides even at pH 6. Similar comparisons of the silver complexes of poly A, poly C, poly I, and poly U, both by potentiometric titration and by spectrophotometry, show that poly I behaves like guanosine and inosine as expected, differing from poly A and poly C. However, poly U behaves like poly I and thus does not resemble uridine in its complexing behavior. There is thus a dichotomy between poly A and poly C on the one hand in silver complexing phenomena, compared with poly U and poly I on the other. When silver(I) is added to systems containing zinc(II) and various polynucleotides, the same dichotomy is noted. Silver(I) inhibits the degradation by zinc(II) of all four polynucleotides, but the degradation of poly I and poly U is prevented virtually completely. Silver(I) alone has no degradative effect on RNA and inhibits, the zinc(II) degradation of RNA. Polynucleotide complexes in which silver(I) and zinc(II) are simultaneously bound to different positions on the macromolecules are postulated as intermediates in the inhibited degradation reactions.     

Involvement of the 3' side of the anticodon loop of yeast tRNATyr in messenger-free binding to ribosomes. An electron-spin resonance study

Electron-spin resonance (ESR) spectra of a nitroxide spin-label attached to residue i6A-37 of yeast tRNATyr were measured in complexes of deacylated tRNATyr with Escherichia coli ribosomes. A Scatchard plot, obtained in the absence of mRNA, indicated strong binding with an association constant of 1 X 10(7) l X mol-1, suggesting the P-site binding. The ESR spectrum of free tRNATyr, characteristic for a rapidly tumbling nitroxide, changes to a spectrum with extensively broadened lines in the ribosome-tRNA complex. The original spectrum can be restored upon long incubations of the complex with an excess of extraneous tRNA. ESR spectra suggest that the spin-label motion is drastically perturbed though not completely blocked in the ribosome-tRNATyr complex. Since ESR spectra of a spin-label attached to the opposite, i.e. 5', side of the anticodon loop are only slightly perturbed by the messenger-free binding to ribosomes [Rodriguez et al. (1980) J. Biol. Chem. 255, 8116-8120], it is concluded that the two sides of the anticodon loop face entirely different environments when bound to the P site, the 3' side being oriented towards the surface of the ribosome, and the other side towards its environment or a large cavity.     

Models of triple-stranded polynucleotides with optimised stereochemistry.

Detailed models are presented for the triple-stranded polynucleotide helices of poly (U)-poly (A)-poly (U) (two forms), poly (U)-poly d (A) -poly (U), poly d(C)-poly d(I)-poly d(C), poly d(T)-polyd(A)-poly d(T) and poly (I)-poly (A)-poly (I). The models were genrated using a computerized, linked-atom procedure which preserves standard bond lengths, bond anglesand sugar ring conformations, constrains the helices to have the pitches and symmetries observed in X-ray diffraction experiments, and optimises the non-bonded interatomic contacts including hydrogen bonds. The possible biological sigificance of such complexes is discussed.     

Interactions of poly-N6-methyladenylic acid and N6-substituted-9-methyladenines with poly-5-bromouridylic acid: a novel base-pairing

The interaction of poly-N6-methyladenylic acid (poly(m6A) with poly-5-bromouridylic acid (poly(BU) was studied by the mixing curve method. A.1 m6A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m6A) to poly(U) is known to occur with 1 m6A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N6-methyl-9-methyladenine (m6m9A), a corresponding monomer of poly(m6A), with poly(BU) was investigated. Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m6A:2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i.r. spectroscopy. Poly (m6A).2poly(BU) and m6m9A.2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N6-(delta 2-isopentenyl)- and N6-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m6m9A.2poly(BU). The melting experiments indicated the Tms to be substantially decreased, compared to the parent unmodified complexes, even though the Tm dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.     

Interaction of poly-5-bromouridylic acid. I. Formation of helical complexes with various adenine and 2-aminoadenine derivatives

Complexes of poly(BU) with various adenine derivatives were investigated by circular dichroism (CD) and absorption spectroscopy. A 1:2 stoichiometry was indicated on CD mixing curves for typical complexes of 9-substituted adenine and 2-aminoadenine derivatives with poly(BU). The CD spectrum of adenosine·2poly(BU) is characterized by well-resolved bands in the range of 210–350 nm. Other adenine derivative–poly(BU) complexes also afford similar CD spectra, while 2-aminoadenine derivative–poly(BU) complexes give quite different spectra. Attempts to assign representative CD spectra were made using the transition of helical poly(BU) and the respective purine polynucleotides. The similarity of the CD spectra suggests that poly(A)·2poly(BU) and adenine derivative–poly(BU) complexes are nearly identical in structure except for the ribose–phosphate linkage. The fact that the uv isosbestic point of adenosine·2poly(BU) falls in close proximity to that of the corresponding polymer complex also supports this conclusion. In the formation of stable helices, the ribose moiety is dispensable in the “strand” of purine. The T_m of 9-methyladenine·2poly(BU) is somewhat higher than that of adenosine·2poly(BU) under equivalent conditions. The T_m difference with the monomer–poly(U) system was found to be about 20°C in 0.4M NaCl–0.02M Na–cacodylate–5 × 10^?4M EDTA (pH 7.0). Further, it was noted that the monomer–poly(BU) complexes are formed even when the T_m is lower than that of self-folded poly(BU).     

Measurements of the time of rotational correlation of bovine serum albumin molecule using iodine and mercury-containing spin-labels

New spin-labels based on iodine and hydrargirum containing imidazolids were approbated on the bovine serum albumin (BSA) molecule. It is shown that all hydrargirum labels are binded to the external SH-group of BSA practically immediately in comparison with earlier known spin-labels based on piperidine with maleimide and iodacetamide groups, requiring some minutes of hours, correspondingly. Rotational correlation times and the character of relative mobility of the spin-label were measured. Values of the rotational correlation times of the protein molecule obtained by hydrargirum containing labels were found in the range of correlation times typical for the BSA molecule and represent at pH 7.0 a rigid stretch ellipsoid of rotation. The result obtained revealed that the relative reorientation nature of spin-labels is essentially different and is taken into account in different values of order parametres S according to model--fast anisotropic rotator on slow isotropic rotator. ESR spectra based on theoretical calculations by means of computer are given.     

Mobility of spin-labelled sulfhydryl sites in excitable tissue

The spin-label 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl was shown to be attached to sulfhydryl groups of the walking leg nerve from the lobster Homarus americanus. Its ESR spectra indicated that it was in a highly immobilized environment. Removal of 90% of the phospholipid by chloroform-methanol extraction had no effect on the degree of immobilization. The ESR spectra of lipid extracted nerves or homogenized nerves showed marked increases in mobility of the spin label when subjected to urea, guanidine·HCl, pH, temperature, proteases, and a smaller shift in response to changes in monovalent cation concentrations. The results are interpreted as a protein conformational shift resulting in increased mobility of the spin-labelled site.     

Conformational change in thrombospondin induced by removal of bound Ca2+. A spin label approach

The effect of removal of Ca2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nano-second time regimes. Removal of bound Ca2+ in TSP by dialyzing spin-labeled TSP from a Ca2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.     

Hydroxyl radical-induced strand break formation of poly(U) in the presence of oxygen: comparison of the rates as determined by conductivity, e.s.r. and rapid-mix experiments with a thiol

The rate of OH radical-induced strand break formation of single-stranded poly(U) in N2O/O2-saturated aqueous solution was studied by measuring the time-dependence of the electrical conductivity following pulse radiolysis. The first half-life of the total conductivity increase depends slightly on pH and the molecular weight and on the dose per pulse. The activation parameters for strand break formation were found to be EA = 52 kJ mol-1 and A = 5 X 10(8) s-1. Similar first half-lives were observed when the decay of peroxyl radicals of poly(U) was measured by e.s.r. under various conditions. This indicates that poly(U)-peroxyl radicals are involved in the rate-determining step of strand break formation. After pulse radiolysis, strand break formation can be inhibited by the addition of dithiothreitol (DTT) in a rapid-mix apparatus. It is postulated that peroxyl radicals of poly(U) react with DTT by formation of hydroperoxides, thereby preventing strand breakage.     

Solution conformation of lactate dehydrogenase as studied by saturation transfer ESR spectroscopy.

Several binary and ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by saturation transfer ESR spectroscopy by means of an active NAD analog, spin-labeled at N6. The mobility of the spin-label depends on the nature of small molecules bound at the remote catalytic end of the coenzyme. The spin-label was found to serve as a reporter group monitoring the conformation of the peptide loop that is folded down over the active cleft in crystals of ternary complexes. The data suggest a fluctuation of the loop between open and closed forms in solution. The structure of the inhibitor molecules has been correlated with their ability to stabilize a more closed conformation of the loop.     

Interactions of poly-N6-methyladenylic Acid and N6-substituted-9-methyladenines with poly-5-bromouridylic Acid: A Novel Base-pairing

Abstract

The interaction of poly-N⁶-methyladenylic acid (poly(m⁶A)) with poly-5-bromouridylic acid (poly(BU)) was studied by the mixing curve method. A 1 m⁶A: 2 BU stoichiometry was clearly indicated over a wide range of ionic strengths at neutral pH, while the binding of poly(m⁶A) to poly(U) is known to occur with 1 m⁶A:1 U. Digestion by nuclease S1 confirmed this stoichiometry, indicating the absence of single strands in a 1:2 mixture. Heating profile analysis and hydroxyapatite column chromatography provided further confirmation of this finding. To determine whether 1:2 stoichiometry holds in a monomer-polymer system, the interaction of N⁶-methyl-9-methyladenine (m⁶m⁹A), a corresponding monomer of poly(m⁶A), with poly(BU) was investigated.

Equilibrium dialysis experiments showed the stoichiometry of the interaction to be 1 m⁶A: 2 BU. Thus, we would describe some structural studies of the above complexes using c.d. and i. r. spectroscopy. Poly (m⁶A)·2poly(BU) and m⁶m⁹A·2poly(BU) are helical and analogous to each other in structure, and the bases in the complexes are all bound by hydrogen-bonding. N⁶-(Δ²-isopentenyl)- and N⁶-allyl-9-methyladenine were also found to form complexes with poly(BU), giving similar c.d. spectra with that of m⁶m⁹A·2poly(BU). The melting experiments indicated the T_ms to be substantially decreased, compared to the parent unmodified complexes, even though the T_m dependence of the polymer complex on salt concentration conforms to the typical triple strand. In the following, the biological significance of this novel pairing will be discussed.     

Vacuum UV CD spectra of homopolymer duplexes and triplexes containing A.T or A.U base pairs.

Vacuum UV circular dichroism (CD) spectra were measured down to 174 nm for five homopolymers, five duplexes, and four triplexes containing adenine, uracil, and thymine. Near 190 nm, the CD bands of poly[d(A)] and poly[r(A)] were larger than the CD bands of the polypyrimidines, poly[d(T)], poly[d(U)], and poly[r(U)]. Little change was observed in the 190 nm region upon formation of the duplexes (poly[d(A).d(T)], poly[d(A).d(U)], poly[r(A).d(T)], poly[r(A).d(U)], and poly[r(A).r(U)]) or upon formation of two of the triplexes (poly[d(T).d(A).d(T)] and poly[d(U).d(A).d(U)]). This showed that the purine strand had the same or a similar structure in these duplexes and triplexes as when free in solution. Both A.U and A.T base pairing induced positive bands at 177 and 202 nm. For three triplexes containing poly[d(A)], the formation of a triplex from a duplex and a free pyrimidine strand induced a negative band centered between 210 and 215 nm. The induction of a band between 210 and 215 nm indicated that these triplexes had aspects of the A conformation.     

Mg2+ and Ni2+ ion effect on stability and structure of triple poly I.poly A.poly I helix

The effects of Mg2+ and Ni2+ ions on the absorption spectra of IMP, single-stranded poly I and three-stranded A2I in solutions with 0.1 M Na+ (pH 7) have been studied. In contrast to Mg2+ ions, the Ni2+ ions affect the absorption spectra of these polynucleotides and IMP. The concentration dependences of the intensity at the extrema in the differential UV spectra suggest that in the region of high Ni2+ concentrations ionic complexes with poly I and A2I are formed, which are characterized by the association constants K'I = 2000 M(-1) and K'A2I = 550 M(-1), respectively. The shape of the DUV spectra prompts the conclusion that these complexes are formed due to the inner-sphere interaction of Ni2+ ions with N7 of poly I and A2I presumably due to the outer-sphere Ni2+-O6 interaction. The formation of the complexes leads to destruction of A2I triplexes. The dependences of the melting temperature (T(m)) of A2I on Mg2+ and Ni2+ concentrations have been measured. The thermal stability is observed to increase at the ionic contents up to 0.01 M Mg2+ and only to 2x10(-4) M Ni2+. At higher contents of Ni2+ ions, T(m) lowers and the cooperativity of A2I melting decreases continuously. In all the cases the melting process is the A2I-->A+I+I (3-->1) transition. According to the "ligand" theory, these effects are generated by the energy-advantageous Ni2+ binding to single-stranded poly I (K'A2I < K'I) and by the greater number of binding sites which appears during the 3-->1 transition and is entropy-advantageous.