NMRDyn: A Program for NMR Relaxation Studies of Protein Association

Self-association is an important biological phenomenon that is associated with many cellular processes. NMR relaxation measurements provide data about protein molecular dynamics at the atomic level and are sensitive to changes induced by self-association. Thus, measurements and analysis of NMR relaxation data can provide structurally resolved information on self-association that would not be accessible otherwise. Here, we present a computer program, NMRdyn, which analyses relaxation data to provide parameters defining protein self-association. Unlike existing relaxation analysis software, NMRdyn can explicitly model the monomer-oligomer equilibrium while fitting measured relaxation data. Additionally, the program is packaged with a user-friendly interface, which is important because relaxation data can often be large and complex. NMRdyn is available from http://research1t.imb.uq.edu.au/nmr/NMRdyn.     

NMR evidence for progressive stabilization of native-like structure upon aggregation of acid-denatured LysN

The acid-denatured form of the protein LysN aggregates reversibly at pH 2.0. The strength of self-association increases with increasing Cl(-) anion concentration. At low concentrations of protein or Cl(-) anion, resonances of denatured LysN are in slow exchange with a minor form of the protein, which shows native-like NMR chemical shifts. The minor native-like resonances increase in intensity with increasing protein concentration, demonstrating that a native-like monomer fold is stabilized on aggregation of the acid-denatured protein. At high concentrations of protein or Cl(-) anion, interconversion between the major and minor resonances appears to shift from slow to intermediate exchange on the NMR timescale. NMR line-broadening is more pronounced for the major resonances of the denatured protein, which show sigmoidal decay curves with increasing Cl(-) concentration. The mid-points of the decay curves for residues in different parts of the molecule are non-coincident. We propose that differences in the NMR line-broadening transitions of individual residues reflect a stepwise stabilization of native-like structure on aggregation, starting with the segments of the protein that form the initial association interface. The resonances of the denatured protein with the greatest sensitivity to self-association correspond roughly to those that are most perturbed in the native protein on binding of the natural substrate tRNA(Lys). This suggests that the hydrophobic surfaces that promote intermolecular misfolding of acid-denatured LysN, may resemble those used for substrate binding by the native protein.     

NMR derived model of GTPase effector domain (GED) self association: relevance to dynamin assembly

Self-association of dynamin to form spiral structures around lipidic vesicles during endocytosis is largely mediated by its 'coiled coil' GTPase Effector Domain (GED), which, in vitro, self-associates into huge helical assemblies. Residue-level structural characterizations of these assemblies and understanding the process of association have remained a challenge. It is also impossible to get folded monomers in the solution phase. In this context, we have developed here a strategy to probe the self-association of GED by first dissociating the assembly using Dimethyl Sulfoxide (DMSO) and then systematically monitoring the refolding into helix and concomitant re-association using NMR spectroscopy, as DMSO concentration is progressively reduced. The short segment, Arg109 - Met116, acts as the nucleation site for helix formation and self-association. Hydrophobic and complementary charge interactions on the surfaces drive self-association, as the helices elongate in both the directions resulting in an antiparallel stack. A small N-terminal segment remains floppy in the assembly. Following these and other published results on inter-domain interactions, we have proposed a plausible mode of dynamin self assembly.     

Inflammasome regulation by adaptor isoforms,ASC and ASCb,via differential self-assembly

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC’s faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.     

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.     

Weak self-association of human growth hormone investigated by nitrogen-15 NMR relaxation

The self-association of human growth hormone(hGH) was investigated using 15N NMR relaxation.The investigation relies on the 15N R1 and R2 relaxation rates and the heteronuclear{1H}-15N NOEs of the backbone amide groups at multiple protein concentrations. It is shown that the rotational correlation time of hGH in solution depends strongly on its concentration, indicating a significant degree of self-association.The self-association is reversible and the monomers in the aggregates are noncovalently linked. Extrapolation of the relaxation data to zero concentration predicts a correlation time of 13.4 ns and a rotational diffusion anisotropy of 1.26 for monomeric hGH, in agreement with the rotational diffusion properties estimated by hydrodynamic calculations. Moreover, the extrapolation allows characterization of the backbone dynamics of monomeric hGH without interference from self-association phenomena, and it is found that hGH is considerably more flexible than originally thought. A concerted least-squares analysis of the 15N relaxations and their concentration dependence reveals that the self-association goes beyond a simple monomer-dimer equilibrium, and that tetramers or other multimeric states co-exist in fast exchange with the monomeric and dimeric hGH at sub-millimolar concentrations. Small changes in the 1H and 15N amide chemical shifts suggest that a region around the C-terminus is involved in the oligomer formation.     

Light chain-dependent self-association of dynein intermediate chain

Dynein light chains are bivalent dimers that bind two copies of dynein intermediate chain IC to form a cargo attachment subcomplex. The interaction of light chain LC8 with the natively disordered N-terminal domain of IC induces helix formation at distant IC sites in or near a region predicted to form a coiled-coil. This fostered the hypothesis that LC8 binding promotes IC self-association to form a coiled-coil or other interchain helical structure. However, recent studies show that the predicted coiled-coil sequence partially overlaps the light chain LC7 recognition sequence on IC, raising questions about the apparently contradictory effects of LC8 and LC7. Here, we use NMR and fluorescence quenching to localize IC self-association to residues within the predicted coiled-coil that also correspond to helix 1 of the LC7 recognition sequence. LC8 binding promotes IC self-association of helix 1 from each of two IC chains, whereas LC7 binding reverses self-association by incorporating the same residues into two symmetrical, but distant, helices of the LC7-IC complex. Isothermal titration experiments confirm the distinction of LC8 enhancement of IC self-association and LC7 binding effects. When all three light chains are bound, IC self-association is shifted to another region. Such flexibility in association modes may function in maintaining a stable and versatile light chain-intermediate chain assembly under changing cellular conditions.     

Human RAD52 exhibits two modes of self-association

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the self-association domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.     

A study of the effect of sodium dodecyl sulfate on bovine β-casein self-association

The effect of low concentrations of sodium dodecyl sulfate on the self-association of β-casein in solution has been reinvestigated at neutral pH by using instrinsic fluorescence measurements, analytical ultracentrifugation, gel filtration chromatography, and the fluorescent properties of the probe, anilinonaphthalene sulfonate. Sodium dodecyl sulfate was found to interact with the protein so that the normal equilibrium between monomers and micellelike polymers was displaced toward polymer formation. At higher concentrations of sodium dodecyl sulfate, the β-casein polymers became smaller while the monomer-polymer equilibrium remained displaced toward polymer formation. It seems likely that there is a limited number of sites on the β-casein molecule that bind sodium dodecyl sulfate strongly. As a consequence of this binding, the balance of electrostatic and hydrophobic forces is altered to increase the degree of self-association at low concentrations of sodium dodecyl sulfate, despite the increase in net negative charge per protein monomer.     

Residue level description of In vivo self-association of Plasmodium falciparum P2

Plasmodium falciparum P2 (PfP2) is a ribosomal stalk protein. It also performs extra ribosomal novel functions that seem to be associated with homo oligomerization . Previous in vitro studies have demonstrated that the protein has a high tendency to self-associate predominantly into an 8-mer. In vitro Heteronuclear Single Quantum Coherence (HSQC) of the pure recombinant protein (rPfP2) and its in-cell (Escherichia coli) HSQC spectrum has very similar features, indicating that the protein intrinsically, both inside the cell and under in vitro conditions, has similar aggregation tendencies. In view of this, we have characterized here the folding and concomitant self-association of rPfP2, using an in vitro dissociation–association strategy. We observed that the residue stretch, (Met31-Leu44) of the rPfP2, mapping to Met1-Leu14 of PfP2 protein acts as a nucleation site for helix formation and subsequent self-association. Further association appears to be driven by hydrophobic and complimentary electrostatic charge interactions on the surfaces formed. One stretch of rPfP2, (Ile97-Ala116), always remains floppy, and this may serve as “hinge” for protein segmental motions. Based on these, we have proposed a possible model for rPfP2 self-association into an 8-mer.     

Glycan Dependence of Galectin-3 Self-Association Properties

Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD). There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.     

NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer

500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.     

Allowance for effects of thermodynamic nonideality in sedimentation equilibrium distributions reflecting protein dimerization

This reexamination of a high-speed sedimentation equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, I(M) 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8 mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of sedimentation equilibrium distributions reflecting reversible protein self-association.     

The role of C-terminal ionic residues in self-association of apolipoprotein A-I

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.     

The terminal phosphate in d(pGpG) reduces self-association

An investigation of the self-association behavior of 2'-deoxy[5'-phosphate-guanylyl-(3'-5')-guanosine] (d(pGpG)) in the presence of Na+ and K+ ions has been carried out by 1H and 31P NMR and FTIR spectroscopy. A comparison has been made of the self- association behavior of d(pGpG) with that of the related dinucleotide d(GpG), which has been shown to form extended structures based on stacked G-tetrads. Chemically, d(pGpG) monomer differs from d(GpG) only by the addition of a phosphate at the 5'-OH of the sugar residue. It was found that the addition of the second phosphate interferes with self-association. A suitable counterion is all that is required by d(GpG) to induce the formation of large super structures, but for d(pGpG) a large excess of salt is needed to produce the same effect. However, once self-association occurs, d(pGpG) forms similar structures to d(GpG) and has nearly the same properties. For both compounds, the K+ ion induces a more stable structure than the Na+ ion. The 31P NMR chemical shift ranges of d(pGpG) were consistent with the reported data for a phosphodiester and terminal phosphate. The small change in the chemical shift of the terminal phosphate with increasing temperature suggests that no major change in the terminal phosphate conformation occurred upon self-association. It was concluded that the terminal phosphate did not result in steric hindrance to self-association, but that interference to self-association was due to electrostatic repulsion effects.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

Stacking interactions of nucleobases: NMR-investigations. II. Self-association of purine-and pyrimidine-derivatives.

The self-association of various purine- and pyrimidine-derivatives in D2O has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model. Among the nucleobases investigated, the adenine-derivatives were found to be most suitable for quantitative determination. A comparison of methylated adenine-derivatives and the pH-dependence of the self-association lead to the conlcusion, that the stacking associates are stabilized by special van der Waals interactions based, essentially, on the polarizability of the pi-electron-system of the assciated molecules.