Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site

The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

In vitro cleavage of Drosophila 2S rRNA by M1 RNA.

Drosophila melanogaster initiator methionine tRNA can adopt an alternative conformation in aqueous solution. In this alternative conformation, the aminoacyl- and the anticodon stems of tRNA are unfolded and then these unfolded regions are used to form extended D- and T-stems, resulting in the formation of two tandemly joined stems and loops. This conformational alternation was recognized then cleaved by the catalytic RNA of Escherichia coli ribonuclease P (M1 RNA). The cleavage occurs within the mature sequence of tRNA. This further processing within mature sequence is called hyperprocessing. During the screening experiments of other conformational changeable D. melanogaster tRNAs by M1 RNA, we incidentally found that M1 RNA also hyperprocessed D. melanogaster 2S rRNA. Kinetic analyses of the hyperprocessing reaction of 2S rRNA by M1 RNA revealed that 2S rRNA could form a homodimer.     

Dynamics and metal ion binding in the U6 RNA intramolecular stem-loop as analyzed by NMR

The U6 RNA intramolecular stem-loop (ISL) is a conserved component of the spliceosome, and contains an essential metal ion binding site centered between a protonated adenine, A79, and U80. Correlated with protonation of A79, U80 undergoes a base-flipping conformational change accompanied by significant helical movement. We have investigated the dynamics of the U6 ISL by analyzing the power dependence of 13C NMR relaxation rates in the rotating frame. The data provide evidence that the conformational transition is centered around an exchange lifetime of 84 micros. The U80 nucleotide displays low internal mobility on the picosecond time-scale at pH 7.0 but high internal mobility at pH 6.0, in agreement with the global transition resulting in the base of U80 adopting a looped-out conformation with increased dynamic disorder. A kinetic analysis suggests that the conformational change, rather than adenine protonation, is the rate-limiting step in the pathway of the conformational transition. Two nucleotides, U70 and U80, were found from chemical shift perturbation mapping to interact with the magnesium ion, with apparent K(d) values in the micromolar to millimolar range. These nucleotides also displayed metal ion-induced elevation of R1 rates, which can be explained by a model that assumes dynamic metal ion coordination concomitant with an induced higher shielding anisotropy for the base 13C nuclei. Addition of Mg2+ shifts the conformational equilibrium toward the high-pH (base-stacked) structure, accompanied by a significant drop in the apparent pK(a) of A79.     

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8

The spike glycoprotein (S) of the murine coronavirus mouse hepatitis virus (MHV) binds to viral murine CEACAM receptor glycoproteins and causes membrane fusion. On virions, the 180-kDa S glycoprotein of the MHV-A59 strain can be cleaved by trypsin to form the 90-kDa N-terminal receptor-binding subunit (S1) and the 90-kDa membrane-anchored fusion subunit (S2). Incubation of virions with purified, soluble CEACAM1a receptor proteins at 37 degrees C and pH 6.5 neutralizes virus infectivity (B. D. Zelus, D. R. Wessner, R. K. Williams, M. N. Pensiero, F. T. Phibbs, M. deSouza, G. S. Dveksler, and K. V. Holmes, J. Virol. 72:7237-7244, 1998). We used liposome flotation and protease sensitivity assays to investigate the mechanism of receptor-induced, temperature-dependent virus neutralization. After incubation with soluble receptor at 37 degrees C and pH 6.5, virions became hydrophobic and bound to liposomes. Receptor binding induced a profound, apparently irreversible conformational change in S on the viral envelope that allowed S2, but not S1, to be degraded by trypsin at 4 degrees C. Various murine CEACAM proteins triggered conformational changes in S on recombinant MHV strains expressing S glycoproteins of MHV-A59 or MHV-4 (MHV-JHM) with the same specificities as seen for virus neutralization and virus-receptor activities. Increased hydrophobicity of virions and conformational change in S2 of MHV-A59 could also be induced by incubating virions at pH 8 and 37 degrees C, without soluble receptor. Surprisingly, the S protein of recombinant MHV-A59 virions with a mutation, H716D, that precluded cleavage between S1 and S2 could also be triggered to undergo a conformational change at 37 degrees C by soluble receptor at neutral pH or by pH 8 alone. A novel 120-kDa subunit was formed following incubation of the receptor-triggered S(A59)H716D virions with trypsin at 4 degrees C. The data show that unlike class 1 fusion glycoproteins of other enveloped viruses, the murine coronavirus S protein can be triggered to a membrane-binding conformation at 37 degrees C either by soluble receptor at neutral pH or by alkaline pH alone, without requiring previous activation by cleavage between S1 and S2.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness

Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). (13)C and (15)N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (?, ψ) torsion angles for three "basis" states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Conformational changes in cubic insulin crystals in the pH range 7-11.

To determine the effect of variations in the charge distribution on the conformation of a protein molecule, we have solved the structures of bovine cubic insulin over a pH range from 7 to 11 in 0.1 M and 1 M sodium salt solutions. The x-ray data were collected beyond 2-A resolution and the R factors for the refined models ranged from 0.16 to 0.20. Whereas the positions of most protein and well-ordered solvent atoms are conserved, about 30% of residues alter their predominant conformation as the pH is changed. Conformational switching of A5 Gln and B10 His correlates with the pH dependence of monovalent cation binding to insulin in cubic crystals. Shifts in the relative positions of the A chain NH2-terminal and B chain COOH-terminal groups are probably due to titration of the A1 alpha-amino group. Two alternative positions of B25 Phe and A21 Asn observed in cubic insulin at pH 11 are similar to those found in two independent molecules of the 2Zn insulin dimer at pH 6.4. The conformational changes of the insulin amino acids appear to be only loosely coupled at distant protein sites. Shifts in the equilibrium between distinct conformational substates as the charge distribution on the protein is altered are analogous to the electrostatically triggered movements that occur in many functional protein reactions.     

Dynamics in the U6 RNA intramolecular stem-loop: a base flipping conformational change

The U6 RNA intramolecular stem-loop (ISL) structure is an essential component of the spliceosome and binds a metal ion required for pre-messenger RNA splicing. The metal binding internal loop region of the stem contains a partially protonated C67-(+)A79 base pair (pK(a) = 6.5) and an unpaired U80 nucleotide that is stacked within the helix at pH 7.0. Here, we determine that protonation occurs with an exchange lifetime of approximately 20 micros and report the solution structures of the U6 ISL at pH 5.7. The differences between pH 5.7 and 7.0 structures reveal that the pH change significantly alters the RNA conformation. At lower pH, U80 is flipped out into the major groove. Base flipping involves a purine stacking interaction of flanking nucleotides, inversion of the sugar pucker 5' to the flipped base, and phosphodiester backbone rearrangement. Analysis of residual dipolar couplings as a function of pH indicates that base flipping is not restricted to a local conformational change. Rather, base flipping alters the alignment of the upper and lower helices. The alternative conformations of the U6 ISL reveal striking structural similarities with both the NMR and crystal structures of domain 5 of self-splicing group II introns. These structures suggest that base flipping at an essential metal binding site is a conserved feature of the splicing machinery for both the spliceosome and group II self-splicing introns.     

A conformational change in bovine beta-lactoglobulin at low pH.

A conformational change at low pH in bovine beta-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate. Both studies show that when the pH of beta-lactoglobulin solutions is altered between 6.5 and 2.0, a rapid change in protein conformation occurs, followed by a slower conformational change. It seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0. The slow changes involve shifts in protein conformation of the region that includes one of the protein tryptophan residues.     

RNA structure analysis using T2 ribonuclease: detection of pH and metal ion induced conformational changes in yeast tRNAPhe.

We describe the use of an enzymic probe of RNA structure, T2 ribonuclease, to detect alterations of RNA conformation induced by changes in Mg2+ ion concentration and pH. T2 RNase is shown to possess single-strand specificity similar to S1 nuclease. In contrast to S1 nuclease, T2 RNase does not require divalent cations for activity. We have used this enzyme to investigate the role of Mg2+ ions in the stabilization of RNA conformation. We find that, at neutral pH, drastic reduction of the available divalent metal ions results in a decrease in the ability of T2 RNase to cleave the anticodon loop of tRNAPhe. This change accompanies an increase in the cleavage of the molecule in the T psi C and in the dihydrouracil loops. Similar treatment of Tetrahymena thermophila 5S ribosomal RNA shows that changes in magnesium ion concentration does not have a pronounced effect on the cleavage pattern produced by T2 RNase. T2 RNase activity has a broader pH range than S1 nuclease and can be used to study pH induced conformational shifts in RNA structure. We find that upon lowering the pH from 7.0 to 4.5, nucleotide D16 in the dihydrouracil loop of tRNAPhe becomes highly sensitive to T2 RNase hydrolysis. This change accompanies a decrease in the relative sensitivity of the anticodon loop to the enzyme. The role of metal ion and proton concentrations in maintenance of the functional conformation of tRNAPhe is discussed.     

Simulation of pH-dependent edge strand rearrangement in human beta-2 microglobulin

Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Analysis of RNA flexibility by scanning force spectroscopy

Scanning force spectroscopy was used to measure the mechanical properties of double stranded RNA molecules in comparison with DNA. We find that, similar to the B–S transition in DNA, RNA molecules are stretched from the assumed A′ conformation to a stretched conformation by applying a defined force (plateau force). The force depends on the G + C content of the RNA and is distinct from that required for the B–S transition of a homologous DNA molecule. After the conformational change, DNA can be further extended by a factor of 0.7 ± 0.2 (S-factor) before melting occurs and the binding of the molecule to the cantilever is finally disrupted. For RNA, the S-factor was higher (1.0 ± 0.2) and more variable. Experiments to measure secondary structures in single stranded RNA yielded a large number of different force-distance curves, suggesting disruption and stretching of various secondary structures. Oriented attachment of the molecules to the substrate, a defined pick-up point and an increased resolution of the instrument could provide the means to analyse RNA secondary structures by scanning force spectroscopy.     

Statistical analysis of RNA backbone

Local conformation is an important determinant of RNA catalysis and binding. The analysis of RNA conformation is particularly difficult due to the large number of degrees of freedom (torsion angles) per residue. Proteins, by comparison, have many fewer degrees of freedom per residue. In this work, we use and extend classical tools from statistics and signal processing to search for clusters in RNA conformational space. Results are reported both for scalar analysis, where each torsion angle is separately studied, and for vectorial analysis, where several angles are simultaneously clustered. Adapting techniques from vector quantization and clustering to the RNA structure, we find torsion angle clusters and RNA conformational motifs. We validate the technique using well-known conformational motifs, showing that the simultaneous study of the total torsion angle space leads to results consistent with known motifs reported in the literature and also to the finding of new ones.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.