We present an approach that speeds up protein solid-state NMR (SSNMR) by 5–20 fold by using paramagnetic doping to condense data-collection time (to ~0.2 s/scan), overcoming a long-standing limitation on slow recycling due to intrinsic 1H T1 longitudinal spin relaxation. By employing low-power schemes under magic-angle spinning at 40 kHz, we show that two-dimensional 13C/13C and 13C/15N SSNMR spectra can be attained for several to tens of nano-moles of β-amyloid fibrils and ubiquitin in just 1–2 days.

It is well established that nitric oxide (•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all •NO-derived adducts (900 pmol/mg protein (45–90 μM)). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics of, DNIC assembly and disappearance from endogenous and exogenous sources of •NO in relation to iron and O2 concentrations. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of •NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic •NO concentrations (50–500 nM). Decreasing the O2 concentration reduced the rate of enzymatic •NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t½ = 80 min) and remained detectable long after the •NO source was removed (>24 h). These results demonstrate that DNIC will be formed under all cellular settings of •NO production and that the contribution of DNIC to the multitude of observed effects of •NO must always be considered.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid ␣-spectrin. The first 20 residues preceding the first helix (helix C) are in a disordered conformation. The subsequent three helices (helices A 1 , B 1 , and C 1 ) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C, the partial domain involved in ␣-and ␤-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C is connected to helix A 1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of ␣-and ␤-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying ␣-and ␤-subunit self-association affinities in their formation of the various spectrin tetramers.Spectrin, a member of the spectrin superfamily and a major protein in the membrane (cyto)skeleton, is ubiquitous among vertebrate tissues, as well as in simple metazoans, implying that spectrin plays a fundamental role in cells (1-3). After first being identified in erythrocytes (4), many distinct spectrin isoforms have since been discovered. In humans, two ␣-spectrin subunits (␣I and ␣II), four ␤-spectrin subunits (␤I, ␤II, ␤III, and ␤IV), and a ␤-H subunit have been sequenced (1). Similar isoforms in mice have also been studied (5). Alternative splicing (e.g. see Ref. 6) adds additional diversity among ␣-and ␤-spectrin isoforms. Many functions of different spectrin isoforms involve interactions with other molecules such as spectrinactin interaction, spectrin-membrane interaction, spectrin-ion channel interaction, etc. (1). Yet some of the most fundamental functions of spectrin involve spectrin "self-association." The activity of spectrin in stabilizing cell-cell contacts and in achieving normal columnar epithelial cell shape requires the formation of tetramers (1,7,8). Spectrin tetramers have been suggested to be cooperatively coupled to membrane assembly (9). Many hereditary hemolytic anemias involve single amino acid mutations in erythrocyte spectrin that destabilize its tetramers, resulting in low levels of spectrin tetramers and high levels of dimers (9 -11). Thus, the tetramerization site is an important functional site for most spectrins.In erythrocyte spectrin (␣I/␤I), ␣-and ␤-spectrin associate with relatively high affinity (nM K d values) at the N-terminal end of the ␤-spectrin and the C-terminal end of the ␣-spectrin (dimer nucleation site) to give ␣␤ heterodimers (12, 1...

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the ␣-subunit and 17 in the ␤-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.DHuman erythrocytes contain a dense, two-dimensional network of spectrin and other proteins that provides support to the lipid bilayer and maintains erythrocyte deformability (1). Spectrin, comprising ␣-and ␤-subunits, plays a critical role in maintaining the architecture and therefore the integrity of the red cell membrane. Many hereditary hemolytic anemias involve spectrin mutations (2-4). Thus, it is important to understand the structural properties of spectrin. The bulk (about 90%) of the primary structure of spectrin comprises repetitive homologous units of approximately 106 amino acids in length. Several other proteins, including brain spectrin (fodrin), dystrophin, and ␣-actinin, also have similar repetitive amino acid units in their sequences and are known as the spectrin superfamily (5).A triple-helical bundle model has been suggested for the 106-amino acid sequence motif in which the three helices are aligned side by side, with the first and third parallel and the intervening second helix antiparallel (6 -8). X-ray diffraction studies of one such sequence motif unit from a non-erythroid spectrin support this model (9). In these x-ray studies, the peptide used was found to form a homodimer containing two triple-helical structures, in which two of the three helices are contributed by one monomer and the remaining helix by the other monomer. It is thought that this peculiar arrangement may be an artifact of crystallization, and the true structure of a single unit may be similar to the earlier suggested triplehelical bundle with a zigzag arrangement of the helices (9, 10). This arrangement aligns the amino-and carboxyl-terminal residues at opposite ends of this triple-helical bundle, and thus sequential motifs are thought to be linked in tandem in intact spectrin, producing a very long rod-shaped molecule, approximately 100 nm in l...

We have studied the effect of the P6-inositol (IHP)-induced change from the quaternary oxy (R) to the deoxy (T) structure in derivatives of human, trout IV, and carp methemoglobins. Addition of IHP to human fluoroand aquomethemoglobin leads to the appearance of the slowly exchanging proton resonance at about -10 ppm from HDO diagnostic of the T structure. This experiment, and the crystallization of aquomethemoglobin + IHP by G. Fermi & M. F. Perutz ((1977) J. Mol. Biol. 114, 421) confirmed that the spectral change in the UV which IHP induces in these compounds can be used as a reliable indicator of the R-*-T transition. Judged by this spectral change, IHP converts all derivatives of carp hemoglobin from the R to the T structure. The pH at which the midpoint of the IHP-induced transition occurs increases with rising spin, being lowest in cyano, intermediate in azido, and highest in thiocyanate and aquomethemoglobin of carp. Conversely the replacement of water by fluoride or thiocyanate as the sixth ligand is unaffected by IHP because all three derivatives are predominantly high spin, but the affinity of azide for carp aquomethemoglobin is reduced 2.7-fold and that of cyanide 3.3-fold by IHP, corresponding to changes in the free energy of binding of 600 and 700 cal/mol heme. Conversion to the T structure of all carp methemoglobin derivatives except the cyanide one is accompanied by large changes in the visible absorption spectra, the most spectacular being that of the nitrite derivative whose color is changed from red to brown. IHP converts all human methemoglobin derivtleme-heme interaction arises from an equilibrium between states which differ in the tertiary structure of the a and 0 subunits and in their quaternary structure in the tetramer. This equilibrium is linked to the stereochemistry at the heme. In deoxyhemoglobin where the tense (T) quaternary structure is dominant, the heme irons are five coordinated and high spin.

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.