Solution conformation of various uridine diphosphoglucose salts as probed by NMR spectroscopy

The solution conformations of uridine diphosphoglucose (UDP-Glc) under a variety of conditions (solvent, ionic strength, various mono- and divalent cations) have been studied by NMR spectroscopy (1H, 13C, 31P, and 25Mg). In the case of divalent cations (Ca2+, Mg2+, Mn2+) the phosphate oxygens are the preferred coordination sites and analysis of the 25Mg linewidths of solutions with various [Mg2+]/[UDP-Glc] ratios, indicates that the 1:1 Mg2+ UDP-Glc complex is the major species. From 13C relaxation data and hydrodynamic theory, it has been demonstrated that under all conditions UDP-Glc adopts a fairly extended overall shape and that magnesium ions lead to a significant increase in the average length of the UDP-Glc molecule as compared to monovalent cations. Thus, one of the roles of the metal ion in enzymic reactions involving nucleotide sugars may be to preorganize the nucleotide sugar.     

Solution structure and dynamics of a heat shock protein assembly probed by hydrogen exchange and mass spectrometry

The solution conformation and dynamics of the 16.9 kDa small heat shock protein from wheat have been studied using a combination of hydrogen/deuterium exchange, proteolytic digestion, and mass spectrometry. At room temperature, HSP16.9 exists as a dodecameric assembly. Regions of HSP16.9 that form extensive and essential intersubunit contacts in the assembly, including residues 1-40 and 131-151, show little or no protection against hydrogen/deuterium exchange after incubation in D(2)O for 5 s. The high levels of hydrogen/deuterium exchange indicate that these regions have experienced large conformational fluctuations in solution, breaking intersubunit contacts and exposing buried amide hydrogens to solvent. When HSP16.9 is pulse labeled for 10 ms, residues 1-40 and 131-151 are substantially more protected than they are after 5 s. Thus, the breaking of intersubunit contacts occurs on a time scale between 10 milliseconds and 5 s. At 42 degrees C, HSP16.9 exists in a suboligomeric form. When the intrinsic temperature dependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 degrees C are identical within experimental error, suggesting that the conformation of individual HSP16.9 subunits is the same in both the dodecameric and subdodecameric forms. Significant protection is seen in regions that form the dimeric interface, suggesting that the stable suboligomeric form is a dimer. Taken together, these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer <--> dimer equilibrium in favor of free dimers. The conformation of the dimers themselves does not appear to be altered with an increase in temperature.     

Weak protein-protein interactions as probed by NMR spectroscopy

Weak protein-protein interactions (PPIs) are fundamental to many cellular processes, such as reversible cell-cell contact, rapid enzyme turnover and transient assembly and/or reassembly of large signaling complexes. However, structural and functional characterizations of weak PPIs have been technically challenging and lagged behind those for strong PPIs. Here, we describe nuclear magnetic resonance (NMR) spectroscopy as a highly effective tool for unraveling the atomic details of weak PPIs. We highlight the recent advances of how NMR can be used to rapidly detect and structurally determine extremely weak PPIs (K(d)>10(-4)M). Coupled with functional approaches, NMR has the potential to look into a wide variety of biologically important weak PPIs at the detailed molecular level, thereby facilitating a thorough view of how proteins function in living cells.     

Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

Cap-dependent ribosome recruitment to eukaryotic mRNAs during translation initiation is stimulated by the eukaryotic initiation factor (eIF) 4F complex and eIF4B. eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The interactions of eIF4E, eIF4A, and eIF4B with mRNA have previously been monitored by chemical- and UV-based cross-linking approaches aimed at characterizing the initial protein/mRNA interactions that lead to ribosome recruitment. These studies have led to a model whereby eIF4E interacts with the 7-methyl guanosine cap structure in an ATP-independent manner, followed by an ATP-dependent interaction of eIF4A and eIF4B. Herein, we apply a splint-ligation-mediated approach to generate 4-thiouridine-containing mRNA adjacent to a radiolabel group that we utilize to monitor cap-dependent cross-linking of proteins adjacent to, and downstream from, the cap structure. Using this approach, we demonstrate interactions between eIF4G, eIF4H, and eIF3 subunits with the mRNA during the cap recognition process.     

Solution conformation and dynamics of a tetrasaccharide related to the LewisX antigen deduced by NMR relaxation measurements

¹H-NMR cross-relaxation rates and nonselectivelongitudinal relaxation times have been obtained at two magnetic fields (7.0and 11.8 T) and at a variety of temperatures for the branchedtetrasaccharide methyl3-O--N-acetyl-galactosaminyl--galactopyranosyl-(14)[3-O--fucosyl]-glucopyranoside (1), an inhibitor of astrocyte growth. Inaddition, ¹³C-NMR relaxation data have also been recorded atboth fields. The ¹H-NMR relaxation data have been interpretedusing different motional models to obtain proton–proton correlationtimes. The results indicate that the GalNAc and Fuc rings display moreextensive local motion than the two inner Glc and Gal moieties, since thosepresent significantly shorter local correlation times. The¹³C-NMR relaxation parameters have been interpreted in termsof the Lipari–Szabo model-free approach. Thus, order parameters andinternal motion correlation times have been deduced. As obtained for the¹H-NMR relaxation data, the two outer residues possess smallerorder parameters than the two inner rings. Internal correlation times are inthe order of 100 ps. The hydroxymethyl groups have also different behaviour,with the exocyclic carbon on the glucopyranoside unit showing the highestS². Molecular dynamics simulations using a solvated systemhave also been performed and internal motion correlation functions have beendeduced from these calculations. Order parameters and interproton distanceshave been compared to those inferred from the NMR measurements. The obtainedresults are in fair agreement with the experimental data.     

Solution phase dynamics of the DNA repair enzyme spore photoproduct lyase as probed by H/D exchange

Spore photoproduct lyase (SPL) catalyzes the repair of the UV lesion spore photoproduct (SP) in a reaction dependent on S-adenosyl-l-methionine (SAM). We have utilized H/D exchange to show that in the presence of SAM, a significant reduction in H/D exchange is observed upon binding SPTpT or undamaged oligonucleotide, indicating a shift of 20 or 10 amide protons, respectively, from a rapidly-exchangable state to a fully-protected conformation. In the absence of SAM, neither the oligonucleotide nor the SPTpT produce a significant perturbation in H/D exchange, indicating SAM is a requisite binding partner. Performing the same experiments in aerobic conditions reduced the magnitude of ligand-induced structural changes, consistent with the importance of the oxygen-sensitive iron–sulfur cluster for SAM and substrate binding.     

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.     

Separation of intramolecular NOE and exchange peaks in water exchange spectroscopy using spin-echo filters

Summary A technique for separating intramolecular NOE and solvent-proton exchange peaks in exchange spectroscopy is demonstrated. This method utilizes the large differences in relaxation and coupling properties of water and macromolecules to separate the two effects. The spin-echo filter consists of a water-frequency selective 90° pulse followed by a spin-echo sequence. If the echo time is sufficiently long, protein resonances (e.g. CH protons) excited by the selective pulse are removed due to their much shorter T₂ values and J-coupling evolution. By combining the filter with exchange spectroscopy (EXSY) or water exchange (WEX) filter experiments, exchange peaks can be selectively observed. In this paper the filter is combined with a modified version of the WEX filter (WEX II filter) with 1D and 2D detection and applied to a zinc finger peptide and to staphylococcal nuclease, allowing estimation of the contribution of intramolecular NOEs to the exchange spectra.To whom correspondence should be addressed.     

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques

The dissociation of the subunits of human adult oxyhemoglobin has been investigated by using steady-state fluorescence anisotropy, multifrequency phase fluorometry, and high hydrostatic pressure. Human hemoglobin obtained by using two purification procedures (bulk preparation by centrifugation or further fractionation using anion-exchange chromatography) was labeled with an extrinsic fluorescent probe, 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS-Cl). The long fluorescence lifetime of this probe allows for the observation of the macromolecular tumbling, and thus provides a method for observing changes in the size of the complex upon subunit dissociation under differing solution conditions of proton and organic phosphate concentration. At pH 7, the dansylated preparations of bulk and fractionated hemoglobin showed a concentration-dependent decrease in the anisotropy which though not identical can only arise from the tetramer to dimer dissociation. We observed primarily the dimer at pH 9 and a small destabilization of the tetramer in the presence of saturating inositol hexaphosphate (IHP). High-pressure experiments allowed for the observation of the dissociation of the hemoglobin dimer into monomers. From these measurements, we estimate the dimer dissociation constant to be between 0.1 and 1 nM. We compare the present results on the subunit affinities in hemoglobin obtained from steady-state and time-resolved fluorescence data with those obtained previously by using gel filtration, sedimentation, and kinetic techniques. These comparisons are indicative of a certain degree of conformational heterogeneity in the hemoglobin preparations.     

A kinetic folding intermediate probed by native state hydrogen exchange

Stopped-flow fluorescence studies on the N-terminal domain of rat CD2 (CD2.d1) have demonstrated that folding from the fully denatured state (U) proceeds via the transient accumulation of an apparent intermediate (I) in a so-called burst phase that precedes the rate-limiting transition leading to the native state (N). A previous pH-dependent equilibrium hydrogen exchange (HX) study identified a subset of amides in CD2.d1 which, under EX2 conditions, exchange from N with free energies greater than or equal to the free energy difference between the N and I states calculated from the stopped-flow data. Under EX1 conditions the rates of HX for these amides tend towards an asymptote that matches the global unfolding rate calculated from the stopped-flow data, suggesting that exchange for these amides requires traversing the N-to-I transition state barrier. Exchange for these amides presumably occurs from exchange-competent forms comprising the kinetic burst phase therefore. To explore this idea further, native state HX (NHX) data have been collected for CD2.d1 under EX2 conditions using denaturant concentrations which span either side of the denaturant concentration where, according to the stopped-flow data, the apparent U and I states are iso-energetic. The data fit to a two-component, sub-global (sg)/global (g) NHX mechanism, yielding Delta G and m value parameters (where the m value is a measure of hydrocarbon solvation). Regression analysis demonstrates that the (m(sg), Delta G(sg)) and (m(g), Delta G(g)) values calculated for this subset of amides correspond with those describing the kinetic burst phase transition. This result confirms the ability of the NHX technique to explore the structural and energetic properties of kinetic folding intermediates.     

Solution conformation of DNA

Optical observations on linear B-form DNA by the method of electric linear dichroism show that the value of the limiting reduced dichroism is molecular weight-dependent, increasing with molecular weight to a limit of about ?1.41 ± 0.02 in aqueous solution. These data and the rotational relaxation times obtained from the decay of the dichroism when the orienting field is instantaneously removed, imply the existence of a non-linear tertiary equilibrium structure for DNA. The data indicate that the essential B-form parameters of the double-stranded DNA are retained in this tertiary structure, and are not consistent with a DNA structure in which the base-pairs have a 34 ° propeller-like twist (Hogan et al., 1978). The interpretation of the dichroism data is supported by a clear demonstration that the magnitude of dichroism change in ethanol corresponds to that expected for the B to A-form structural transition as determined from X-ray diffraction. We propose that the tertiary structure of B-DNA is a helical coil and suggest the limits of the structural parameters of the coil consistent with the observations.     

Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines

Glycolipid antigen was examined by radioimmuno-thin-layer chromatography (RITLC), which is a combination of a thin-layer chromatography and radioimmunoassay. In this way Forssman antigen was studied in seven carcinoma cell lines. The usual Forssman antigen with a ceramide pentasaccharide structure was detected in cell lines of a gastric cancer and a breast cancer. In addition another glycolipid with slower mobility on thin-layer chromatography and with Forssman reactivity was found in cell lines of three gastric cancers and one lung cancer.     

Lateral ligand-receptor interactions on membranes probed by simultaneous fluorescence-interference detection

We describe an experimental approach for studying ligand-receptor interactions in the plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFNalpha2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e., association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium, and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFNalpha2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.     

Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting

Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed.