Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy.

The arrival of very high field magnets and cryogenic circuitries, and the development of relaxation-optimized pulse sequences have added powerful tools for increasing sensitivity and resolution in NMR studies of biomacromolecules. The potential of these advances is not fully realized in practice, however, since current experimental protocols do not permit sufficient data sampling for optimal resolution in the indirect dimensions. Here we analyze quantitatively how increasing resolution in indirect dimensions affects the S/N ratio and compare this with currently used sampling routines. Optimal resolution would require sampling up to approximately 3 R (2)(-1), and the S/N reaches a maximum at approximately 1.2 R (2)(-1). Currently used data acquisition protocols rarely sample beyond 0.4 R (2)(-1), and extending evolution times would result in prohibitively long experiments. We show that a general solution to this problem is to use non-uniform sampling, where only a small subset of data points in the indirect sampling space are measured, and possibly different numbers of transients are collected for different evolution times. Coupled with modern methods of spectrum analysis, this strategy delivers substantially improved resolution and/or reduced measuring times compared to uniform sampling, without compromising sensitivity. Higher resolution in the indirect dimensions will facilitate the use of automated assignment programs.

De novo backbone and sequence design of an idealized alpha/beta-barrel protein: evidence of stable tertiary structure.

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm.

Aromatic Ring Currents at a Protein Surface: Use of (1)H-NMR Chemical Shifts to Refine the Structure of a Naked ß Sheet.

Abstract The naked ß sheet, a newly recognized motif of protein structure, exhibits ordered surfaces in the absence of a conventional hydrophobic core. A model is provided by an archaeal Zn ribbon homologous to eukaryotic RNA polymerase II subunit 9 (RPB9). This subunit, which regulates transcriptional start-site selection and downstream pausing, contains Zn(2+)-binding motifs similar to those of general transcription factors TFIIB and TFIIS. Interestingly, distance-geometry yields two models of the archaeal Zn ribbon differing in the orientation of a conserved tyrosine side chain on the well-ordered surface of the naked ß-sheet. The models are equally consistent with conventional restraints and otherwise contain indistinguishable structural features, including a tetrahedral Cys(4) Zn(2+)-binding sites, four antiparallel ß-strands, and disordered loop. Due to the change in tyrosine orientation and correlated changes in the configuration of neighboring side chains, the two models predict inequivalent patterns of aromatic ring-current shifts. The observed secondary shifts of adjoining resonances are shown to be consistent with one model but not the other. In the consistent model the surface of the ß-sheet contains successive aromatic edge-to-face contacts in accord with semi-classical and ab initio potentials. We speculate that the aromatic-rich surface of the hyperthermophilic RPB9 domain contributes its thermodynamic stability and provides a nucleic-acid-binding site in the eukaryotic and archaeal transcriptional machinery. The present study demonstrates how the reduced dimensionality of a surface can lead to ambiguities in the interpretation of nuclear Overhauser enhancements. The results illustrate the utility of chemical shifts at such a surface in the cross-validation of a high-resolution solution structure.

Temperature dependence of switching of the bacterial flagellar motor by the protein CheY(13DK106YW).

The behavior of the bacterium Escherichia coli is controlled by switching of the flagellar rotary motor between the two rotational states, clockwise (CW) and counterclockwise (CCW). The molecular mechanism for switching remains unknown, but binding of the response regulator CheY-P to the motor component FliM enhances CW rotation. This effect is mimicked by the unphosphorylated double mutant CheY13DK106YW (CheY**). To learn more about switching, we measured the fraction of time that a motor spends in the CW state (the CW bias) at different concentrations of CheY** and at different temperatures. From the CW bias, we computed the standard free energy change of switching. In the absence of CheY, this free energy change is a linear function of temperature (. Biophys. J. 71:2227-2233). In the presence of CheY**, it is nonlinear. However, the data can be fit by models in which binding of each molecule of CheY** shifts the difference in free energy between CW and CCW states by a fixed amount. The shift increases linearly from approximately 0.3kT per molecule at 5 degrees C to approximately 0.9kT at 25 degrees C, where k is Boltzmann's constant and T is 289 Kelvin (= 16 degrees C). The entropy and enthalpy contributions to this shift are about -0. 031kT/ degrees C and 0.10kT, respectively.

Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL-12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL-12 antagonist. To study the interaction between (p40)2 and the known IL-12 receptor (IL-12R) subunits, IL-12Rbeta1 and IL-12Rbeta2, we directly measured the binding activity of mouse (p40)2 to ConA-activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I-labeled (p40)2. To elucidate which of the IL-12R subunits binds mouse (p40)2, binding studies of mouse 125I-labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL-12Rbeta1 and/or mouse IL-12Rbeta2 were carried out. Mouse IL-12Rbeta1 bound mouse 125I-labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL-12Rbeta2 bound mouse 125I-labeled (p40)2 very poorly. These data demonstrate that similar to IL-12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL-12Rbeta1 is responsible for mediating the specific binding of mouse (p40)2.

Artifacts in sensitivity-enhanced HSQC.

Proton-proton coupling can generate artifacts in sensitivity-enhanced HSQC spectra. These artifacts appear as cross-peaks involving remote protons. They are caused by relayed coherence transfer during the back-transfer portion of the pulse sequence. We present a product operator analysis of artifact formation and experimental results which demonstrate that the magnitude of these artifacts can exceed 10% of the main peak.

Modeling physical health and functional health status: the role of combat exposure, posttraumatic stress disorder, and personal resource attributes.

This study examined associations of combat exposure and posttraumatic stress disorder (PTSD) with physical health conditions and also incorporated hardiness and social support as mediators and functional health status as an outcome. Data were derived from 1,632 male and female Vietnam veterans who participated in the National Vietnam Veterans Readjustment Study. Path analysis revealed that hardiness and social support operated primarily as intermediary variables between combat exposure and PTSD, and PTSD emerged as the pivotal variable explaining physical health conditions and functional health status. Gender-based differences in means and patterns of associations among variables were found. The results stress the importance of assessing trauma in clinical settings as a meaningful determinant of health outcomes.

Distributed parallel processing for multidimensional maximum entropy reconstruction.

We have developed a two-dimensional maximum entropy spectrum reconstruction program designed to run in parallel on workstation clusters. Test reconstructions of planes extracted from a three-dimensional NMR data set indicate that the parallel speedup is nearly equal to the number of processors provided that the individual processors have comparable performance and that there are at least as many planes as processors. The program also works well in a typical laboratory setting consisting of heterogeneous workstations.

The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2. Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled. Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens. However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology. Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease. On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma.

Quantification of maximum-entropy spectrum reconstructions.

Maximum-entropy spectrum reconstruction derives much of its power from its nonlinearity. This nonlinearity causes difficulties in several contexts, however, including computation of multidimensional spectra and quantification of reconstructed spectra. We describe two methods for avoiding these difficulties: a "Constant-lambda" algorithm for performing row-wise reconstructions, which uses a fixed weighting of the entropy and the experimental constraint in the objective function, and in situ error analysis, for calibrating the nonlinearity. These methods are applied to data from quantitative J-correlation and relaxation experiments.

Interleukin-12 antagonist activity of mouse interleukin-12 p40 homodimer in vitro and in vivo.

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Evidence for multiple sites of interaction between IL-12 and its receptor.

We have previously described the identification of a protein, now designated IL-12R beta 1, that binds 125I-huIL-12 with a Kd of about 10 nM, corresponding to the low affinity 125I-huIL-12 binding sites seen on PHA-activated human lymphoblasts. Using expression cloning techniques, we have recently identified an additional IL-12-binding protein subunit, IL-12R beta 2, which binds 125I-huIL-12 with a Kd of about 5 nM when expressed alone in COS-7 cells. Coexpression of IL-12R beta 1 and IL-12R beta 2 in COS-7 cells results in formation of two classes of 125 I-huIL-12-binding sites with Kds of about 50 pM and 5 nM. Mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to human IL-12R beta 1, but did not inhibit binding to human IL-12R beta 2. In contrast, anti-huIL-12 monoclonal antibody 20C2, which does not block 125I-huIL-12 binding to human IL-12R beta 1, completely blocked binding to human IL-12R beta 2. These results demonstrate that two classes of IL-12 inhibitors, one that primarily blocks IL-12/IL-12R beta 1 interaction (e.g., mo(p40)2), and one that primarily blocks IL-12/IL-12R beta 2 interaction (e.g., 20C2), can be identified.

Structure and dynamics of a protein assembly. 1H-NMR studies of the 36 kDa R6 insulin hexamer.

The structure and dynamics of the R6 human insulin hexamer are investigated by two- and three-dimensional homonuclear 1H-NMR spectroscopy. The R6 hexamer, stabilized by Zn2+ and phenol, provides a model of an allosteric protein assembly and is proposed to mimic aspects of receptor recognition. Despite the large size of the assembly (36 kDa), its extreme thermal stability permits high-resolution spectra to be observed at 55 degrees C. Each spin system is represented uniquely, implying either 6-fold symmetry or fast exchange among allowed protomeric conformations. Dramatic changes in chemical shifts and long-range nuclear Overhauser enhancements (NOEs) are observed relative to the spectra of insulin monomers. Complete sequential assignment is obtained and demonstrates native secondary structure with distinctive R-state N-terminal extension of the B-chain alpha-helix (residues B1 to B19). The distance-geometry structure of an R-state promoter is similar to those of R6 crystal structures. Specific long-range intra- and intersubunit NOEs, assigned by stepwise analysis of engineered insulin monomer and dimers, demonstrate that tertiary and quaternary contacts are also similar. Although the hexamer is well-ordered in solution, binding of phenol to an internal cavity occurs within milliseconds, implying the existence of "gatekeeper" residues whose flexibility provides a portal of entry and release. Changes in 1H-NMR chemical shifts on hexamer assembly are readily rationalized by analysis of aromatic ring-currents and provide sensitive probes for sites of protein-protein interaction and phenol binding. Our results provide a foundation for the interaction and phenol binding. Our results provide a foundation for the studies of insulin analogues (such as "designed" insulins of therapeutic interest) under conditions of clinical formulation and for the investigation of the effects of protein assembly on the dynamics of individual protomers.

Biological function and distribution of human interleukin-12 receptor beta chain.

We previously described the cloning of a cDNA encoding an interleukin-12 receptor (IL-12R) subunit, designated beta, that bound IL-12 with low affinity when expressed in COS cells. We now report that a pair of monoclonal antibodies (mAb), 2B10 and 2.4E6, directed against different epitopes on the IL-12R beta chain, when used in combination, strongly inhibited IL-12-induced proliferation of activated T cells, IL-12-induced secretion of interferon-gamma by resting peripheral blood mononuclear cells (PBMC), and IL-12-mediated lymphokine-activated killer cell activation. The mAb had no effect on lymphoblast proliferation induced by IL-2, -4, or -7. Thus, the IL-12R beta chain appears to be an essential component of the functional IL-12R on both T and natural killer (NK) cells. We previously observed that high affinity IL-12R were expressed on activated T and NK cells, but not B cells. Studies using flow cytometry and reverse transcription-polymerase chain reaction analysis showed that IL-12R beta chain was expressed on several human T, NK, and (surprisingly) B cell lines, but not on non-lymphohematopoietic cell lines. The Kit225/K6 (T cell) and SKW6.4 (B cell) lines were found to express the greatest amounts of IL-12R beta chain (800-2500 sites/cell); however, Kit225/K6 but not SKW6.4 cells bound IL-12. Similar to SKW6.4 B cells, activated tonsillar B lymphocytes expressed IL-12R beta chain but, consistent with previous results, did not display detectable IL-12 binding. Likewise, up to 72% of resting PBMC from normal volunteer donors expressed IL-12R beta, but did not bind measurable amounts of IL-12. These results indicate that expression of IL-12R beta is essential, but not sufficient, for expression of functional IL-12R. We speculate that expression of functional, high-affinity IL-12R may require the presence of a second subunit that is more restricted in its expression than IL-12R beta.

Solution structure of LSIII, a long neurotoxin from the venom of Laticauda semifasciata.

We report the sequence-specific proton assignments and solution structure of the long neurotoxin LSIII from the venom of Laticauda semifasciata determined by two- and three-dimensional 1H NMR. Input for structure calculations consisted of 497 NOE-derived distance restraints and 45 dihedral angle restraints obtained from J couplings. A two-particle-per-residue representation of protein structure was used to generate 200 initial structures which were then subjected to all-atom refinement by simulated annealing. Twenty-three final structures consistent with the experimental restraints were obtained; the average atomic RMS difference between the individual structures and the mean structure was 0.82 A for the backbone heavy atoms and 1.3 A for all heavy atoms (residues 1-26, 37-60). The main elements of regular secondary structure are a three-stranded antiparallel beta-sheet and three finger-like loops protruding from a globular core, consistent with previously reported structures of long neurotoxins. The end of the prominent loop II, which is involved in binding to acetylcholine receptor, is disordered relative to the rest of the molecule. A novel finding of this study is that the loop has a well defined local structure; this and other observations suggest this region moves as a rigid body. We propose that this motion is a heretofore unrecognized general feature of long neurotoxins, with specific consequences for binding to the acetylcholine receptor.

Interleukin-12 an integral cytokine in the immune response.

Interleukin 12 (IL-12) is a heterodimeric cytokine that is produced primarily by antigen-presenting cells and plays a primary role in the induction of cell-mediated immunity. This function is promoted by the IL-12 induced production of interferon-gamma (IFN-gamma) from both resting and activated NK and T cells, by the proliferative activity of IL-12 on activated NK and T cells, by enhancing the cytotoxic activity of NK cells, and by supporting cytotoxic T lymphocyte generation. IL-12 and IL-12-induced IFN-gamma promote the development of naive T cells into Th1 cells and the proliferation and IFN-gamma secretion by differentiated Th1 cells in response to antigen. IL-12 has been found to exhibit many of these activities in vivo, as well as in vitro, and thus IL-12 plays an important role in both innate resistance and antigen-specific adaptive immunity to intracellular bacterial, fungal, and protozoan pathogens. Due to its effects on T cells, recombinant IL-12 has been shown to have therapeutic activity in a variety of mouse tumor and infectious disease models and is being evaluated in clinical trials in human cancer patients. IL-12 also appears to play a role in the genesis of some forms of immunopathology, including endotoxin-induced shock and some autoimmune diseases associated with aberrant Th1 activity. Therefore, IL-12 antagonists may also have therapeutic potential in the treatment of auto immune disorders.

Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist.

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.