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At least 19 recordsLinked to original sources
PubMed2024

Regulating IL-2 Immune Signaling Function Via A Core Allosteric Structural Network.

Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2's pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2's therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and minor state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkine's core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles.

Interleukin-2↗
PubMed1992

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation.

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).

Adenosine Triphosphate↗
PubMed1992

FTIR spectroscopic studies of the conformation and amide hydrogen exchange of a peptide model of the hydrophobic transmembrane alpha-helices of membrane proteins.

The conformation and amide hydrogen exchangeability of the hydrophobic peptide Lys2-Gly-Leu24-Lys2-Ala-amide were studied by Fourier transform infrared spectroscopy. In these studies information on the secondary structure of the peptide was obtained from an examination of the contours of both the amide I and amide II absorption bands. The conformationally sensitive amide I and amide II regions of the infrared spectra suggest that the peptide is predominantly alpha-helical and that it contains some non-alpha-helical structures which are probably in an extended conformation. Studies of the exchangeability of the amide protons of the peptide indicate that there are two populations of amide protons which differ markedly with respect to their exchangeability with the bulk solvent phase, whether the peptide is dissolved in methanol or dispersed in hydrated lipid bilayers. One population of amide protons is very readily exchangeable, and our data suggest that it arises primarily but not exclusively from the extended regions of the peptide. The other population exchanges very slowly with the bulk solvent and appears to originate entirely from the alpha-helical domain of the peptide. This latter population is virtually unexchangeable when the peptide is dispersed in hydrated phosphatidylcholine bilayers but can be largely exchanged when the peptide is solubilized with methanol. We suggest that this slowly exchanging population of amide protons arises from the central part of the hydrophobic polyleucine core which forms a very stable alpha-helix that would be deeply buried in the hydrophobic domain of hydrated lipid bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1977

Substrate-induced deactivation of penicillinases. Studies of beta-lactamase I by hydrogen exchange.

The conformational motility of beta-lactamase I from Bacillus cereus was studied by hydrogen exhange. The time course of the isotopic replacement of peptide hydrogen atoms was followed by 'exchange-in' or 'exchange-out' experiments. Many of the substrates for this enzyme that have o-substituted aromatic or heterocyclic side chains (e.g. methicillin or cloxacillin) are known to effect a decrease in enzymic activity ('substrate-induced deactivation'). There was a marked discontinuity in the exchange-out curve when methicillin or cloxacillin was diffused into the enzyme solution. About one-half of the hydrogen atoms that were probed were affected by the presence of these substrates, and the change in the reactivity of the hydrogen atoms was also large. Substrates that do not bring about deactivation (benzylpenicillin and cephalosporin C) do not affect the hydrogen exchange, nor do reversible competitive inhibitors such as the penicilloic acid or penilloic acid. On the other hand, Zn2+ ions do affect the hydrogen exchange; their effect is similar to that of methicillin or cloxacillin.

Cloxacillin↗
PubMed1975

Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (Kunitz). Solvent accessibility in the folded conformation.

The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, THE CONTRIBUTION OF MAJOR, REVERSIBLE PROTEIN UNFOLDING To the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but congruent to 8 protons can be observed to exchange with E*app congruent to 27 kcal/mol. This implies that all but congruent to 8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30 degrees, approximating STI structure by a sphere of radius = 18 A. These calculations indicate an average of congruent to 4 water molecules in the shell from 13 to 16 A. from the center of the molecule, while less than 1 water molecule is indicated in the innermost 13 A. These calculations also suggest that there are congruent to 190 water molecules associated with the outermost 1.5-2 A of the sphere. While these values are consistent with a hydrophobic region in the central protein matrix, they indicate more solvent accessibility in the outer 1/3 of the molecule than the static accessibility estimates made from X-ray coordinates. Our results suggest that any protein movements or fluctuations responsible for solvent accessibility in proton exchange processes are localized in the outer regions of the globular structure.

Binding Sites↗
PubMed1975

Hydrogen-exchange study of the conformational stability of human carbonic-anhydrase B and its metallocomplexes.

In the range of pH 4.6--8.8, 25 degrees C, the apoenzyme of carbonic anhydrase B shows no evidence of any gross conformational changes, as studied by the hydrogen-deuterium exchange method. At pH 4.6 the addition of Co(II), Cd(II) or Mn(II) to the apoenzyme results in a destabilization of the native protein conformation, but in the range of pH 5.5--8.8 these metal ions, and Zn(II), slightly increase the conformational stability of the protein, in so far as they reduce the probability phi of solvent exposure of the peptide groups. In comparison with other proteins studied, native carbonic anhydrase is characterized by a rather compact conformation; for half of the peptide groups the probability of solvent exposure is less than 10(-4), corresponding to changes in standard free energy larger than 5.5 kcal mol-1 (23 kJ mol-1) following the conformational transitions by which these groups are exposed to the solvent.

Apoenzymes↗
PubMed1978

The conformation of membrane-bound and detergent-solubilised bovine rhodopsin. A comparative hydrogen-isotope exchange study.

The conformations of the intrinsic membrane protein, rhodopsin, in its membrane-bound and detergent-solubilised states have been compared by hydrogen isotope exchange measurements. The infrared peptide exchange data show that the highly hydrophobic nature of rhodopsin is conserved in the presence of the two detergents used: Cemulsol LA 90 and Ammonyx LO. Only about 50% of the peptide hydrogens exchange under conditions where about 80% would exchange in most soluble proteins. The conformational stability of rhodopsin in these two detergents is also demonstrated by the similarity of the tritium exchange-out kinetics and the infrared amide I band frequencies for both membrane-bound and detergent-solubilised rhodopsin. Upon illumination of rhodopsin (bleaching) in the presence of detergents, the hydrogen exchange rates are greatly increased and shifts in the amide I band frequencies are observed, indicative of a large conformation change. No such change occurs upon bleaching membrane-bound rhodopsin. We conclude that the conformation of rhodopsin is not altered by solubilisation in non-ionic detergents. However, in agreement with previously published results, bleached rhodopsin is stabilised by the membrane but does not retain a native conformation in these detergents.

Animals↗
PubMed1992

Effects of glycosylation on protein conformation and amide proton exchange rates in RNase B.

Assignment of most of the proton NMR resonances of bovine pancreatic RNase B has been achieved using standard NMR techniques and by comparison with the published assignments for RNase A. A comparison of the NMR spectra of RNase B with RNase A shows that glycosylation of the enzyme has little overall effect on the conformation of the protein in solution. Comparisons of hydrogen-deuterium solvent exchange rates for the NH protons of RNase A and RNase B were made using two-dimensional 1H correlation spectroscopy. In the case of the glycosylated enzyme the exchange rates decreased for the NH protons of residues 9-14, 23-24, 32, 34-35, 39-40, 43-44, 48-49, 60, 71, 75-76, 80, 83-85, 100-101, 107, 111 and 122, relative to the unglycosylated RNase A. These results are consistent with the presence of the oligosaccharide inducing enhanced global dynamic stability and consequent changes to the unfolding equilibrium of the enzyme. The enhanced stability is observed not only for residues in the vicinity of the glycosylation site, asparagine-34, but also at residues remote from this site, as much as 30 A away.

Amides↗
PubMed1978

Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange.

Catalysis-linked conformational transitions of aspartate aminotransferase (cytosolic isoenzyme from pig heart; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been probed by infrared spectrophotometric measurement of hydrogen-deuterium exchange. In the unliganded pyridoxal form of the enzyme at pH 6.0 and 20 degrees, 43% of the total 411 peptide hydrogens per subunit exchange within the first 10 min. An additional 9% exchange slowly in the following time period to 360 min. A quite similar exchange curve is obtained with the pyridoxamine form of the enzyme, indicating close correspondence in conformation of both unliganded forms of the enzyme. Formation of a nonproductive adsorption complex of the pyridoxal enzyme with 2-oxoglutarate or of the pyridoxamine enzyme with glutamate alters the exchange characteristics only slightly. In contrast, the formation of an equilibrium mixture of the covalent transamination intermediates, which occurs in the silultaneous presence of the amino acid and the keto acid substrate, results in a marked retardation of hydrogen exchange, reflecting a substantial tightening of the structure of the enzyme. The exchange reactions of at least 26 peptide hydrogens per subunit (6% of the total) are retarded by a factor of 6 on the average. The occurrence of such syncatalytic conformational changes reflects energetic coupling of the covalency changes at the active site with conformational changes of the macromolecular protein matrix that may contribute to optimizing the free energy profile of enzymic transamination.

Animals↗
PubMed1975

Chloroplast membranes and coupling factor conformations.

The demonstrated role of proton translocation and resulting electrochemical activity gradients (protonmotive force) in ATP synthesis by chloroplasts is noted. Evidence for the participation of conformational changes in the terminal ATPase (coupling factor, or CF1) is reviewed. Hydrogen exchange into ordinarily cyptic groups of the molecule occurs only when the subtending membranes are put under the stress of a protonmotive force. Since up to 100 hydrogen atoms per mole are involved in the energy-dependent exchange the conformational change permitting tham access to the medium must be a major one. Chemical reagents are beginning to be used to attack groups on CF1 that are exposed only when the membranes are energized. N-ethylmaleimide binds covalently, sulfate causes as yet unspecified damage, and permanganate leads to oxidative damage to CF1 under energized conditions. The last two reagents are analogues of phosphate, and ADP must be added for them to inhibit. On the basis of this and other differences between the conditions needed for inhibition by permanganate or sulfate, and that by N-ethylmaleimide or the hydrogen exchange, a somewhat complex scheme involving several successive or alternative conformations of CF1 can be postulated. Questions are raised as to the way in which a conformational change in a bound protein could be caused by a proton activity gradient across its supporting membrane, and as to whether the altered conformations might constitute a part of the energy transformations leading to ATP synthesis.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Mechanism of coupling of ion transport and ATP hydrolysis in the Na-pump].

A generalized scheme of the reaction pathways during activation of the Na,K-ATPase by sodium and potassium ions and a relevant molecular model of the Na-pump are proposed. The model suggests light and heavy enzyme subunits possessing cavities with ion exchange sites. The cavities are of limited size and can contain only 3 sodium or 2 potassium ions. Free energy of ATP hydrolysis is expended on the formation of a special transient nonequilibrium enzyme conformation. In this conformation ion exchange between the subunit cavities becames possible. Na-pump operates as an enthropy machine: the ion movement across the membrane is provided by thermal oscillations of the subunits.

Binding Sites↗
PubMed1992

A study of hydrogen exchange of monoclonal antibodies: specificity of the antigen-binding induced conformational stabilization.

Amide-hydrogen exchange of three anti-yeast iso-1-cytochrome-c IgG monoclonal antibodies and the Fab, prepared from one of them, were studied by infrared spectrophotometry in the presence and absence of the deuterated immunogen and evolutionarily related species (the deuterated immunogen contained a population of a dimer. Each subunit of the dimer appeared to bind to the antibodies in a manner similar to the monomer). The number of hydrogens of the antibodies whose exchange was suppressed on binding to the immunogen was found to exceed that estimated for the residues shielded by the immunogen. Analysis of the data suggests that such suppression of hydrogen exchange occurs mainly for the Fab domains, but not for the Fc. One of the antibodies showed two distinct classes of amide-hydrogens. Class-1 hydrogens (approx. 36/site) exchange faster than class 2 (approx. 37/site). The exchange of class-1 hydrogens was suppressed by binding to the immunogen, but not to the evolutionarily related species. The exchange of class-2 hydrogens was suppressed by binding to the evolutionarily related species, as well as to the immunogen. Thus, the suppression of exchange of class-1 hydrogens appears to occur by some kind of conformational stabilization, the mechanism of which differentiates between the deuterated immunogen and the evolutionarily related species. Evidence suggests that the trans-interactions of the Fab domains may modulate the hydrogen exchange. If it is assumed that the antigen-binding strengthens the trans-interactions in such a way that the exchange of the slower exchanging hydrogens is suppressed, this could explain the suppression of exchange of class-2 hydrogens.

Amides↗
PubMed1992

Proton resonance assignments and three-dimensional solution structure of the ragweed allergen Amb a V by nuclear magnetic resonance spectroscopy.

Essentially complete assignment of the proton resonances in the allergenic protein Amb a V has been made by analysis of two-dimensional NMR experiments. Conformational constraints were obtained in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb a V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a small segment of antiparallel beta-sheet, and several loops. A hydrophobic core exists at the interface of the alpha-helix and beta-sheet. The derived structure accounts for the several anomalous proton chemical shifts that are observed. The structure determined here for Amb a V is topologically similar to the structure determined previously for the homologous allergenic protein Amb t V [Metzler, W. J., Valentine, K., Roebber, M., Friedrichs, M. S., Marsh, D., & Mueller, L. (1992) Biochemistry 31, 5117-5127]; however, significant differences exist in the packing of side chains in the hydrophobic core of the molecules. Comparison of the detailed structural features of these two proteins will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes.

Allergens↗
PubMed1992

Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.

Amino Acid Sequence↗
PubMed2026

Multidimensional Protein Corona Analysis Toward Predictive Nano-Bio Interface Design.

Nanoparticles entering biological fluids are rapidly coated by proteins and other biomolecules, converting their synthetic surfaces into biologically active nano-bio interfaces. These coronas regulate colloidal stability, immune recognition, cellular uptake, biodistribution, pharmacokinetics, cargo delivery, and toxicity. Yet a protein list obtained by mass spectrometry captures only part of this interface. Corona identity and function are also shaped by protein organization, binding stability, exchange dynamics, conformational changes, and molecular accessibility. Here, we discuss recent progress in protein corona isolation and analysis from a question-oriented analytical perspective, with emphasis on how centrifugation, magnetic recovery, affinity- or chemistry-enabled capture, chromatography, filtration, and field-flow fractionation (FFF) influence the fidelity, integrity, and comparability of recovered coronas. We then examine how proteomic profiling can be integrated with binding measurements, interfacial structural analysis and functional validation to distinguish descriptive corona signatures from biologically meaningful mechanisms. We further consider how biofluid composition, disease state, tissue interfaces and cellular environments remodel corona identity, presentation, and bioactivity. Finally, we argue that standardized reporting, computational modeling, and AI-enabled approaches are essential for converting protein corona datasets into reproducible and predictive knowledge that can guide the design of drug delivery systems and precision nanomedicines.

Protein Corona↗
PubMed1992

Backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The extent of rapid (picosecond) backbone motions within the glucocorticoid receptor DNA-binding domain (GR DBD) has been investigated using proton-detected heteronuclear NMR spectroscopy on uniformly 15N-labeled protein fragments containing the GR DBD. Sequence-specific 15N resonance assignments, based on two- and three-dimensional heteronuclear NMR spectra, are reported for 65 of 69 backbone amides within the segment C440-A509 of the rat GR in a protein fragment containing a total of 82 residues (MW = 9200). Individual backbone 15N spin-lattice relaxation times (T1), rotating-frame spin-lattice relaxation times (T1 rho), and steady-state (1H)-15N nuclear Overhauser effects (NOEs) have been measured at 11.74 T for a majority of the backbone amide nitrogens within the segment C440-N506. T1 relaxation times and NOEs are interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) characterizing internal motions in each backbone amide using an optimized value for the correlation time for isotropic rotational motions of the protein (tau R = 6.3 ns). Average S2 order parameters are found to be similar (approximately 0.86 +/- 0.07) for various functional domains of the DBD. Qualitative inspection as well as quantitative analysis of the relaxation and NOE data suggests that the picosecond flexibility of the DBD backbone is limited and uniform over the entire protein, with the possible exception of residues S448-H451 of the first zinc domain and a few residues for which relaxation and NOE parameters were not obtained. in particular, we find no evidence for extensive rapid backbone motions within the second zinc domain. Our results therefore suggest that the second zinc domain is not disordered in the uncomplexed state of DBD, although the possibility of slowly exchanging (ordered) conformational states cannot be excluded in the present analysis.

Amides↗
PubMed1975

The interaction of adenosinetriphosphate and inorganic phosphate with the sodium pump in red cells.

1. An increase in the intracellular concentration of inorganic phosphate (Pi) reduces the rate of the Na:K exchange catalysed by the Na pump in red cells. The inhibitory effect of Pi is exerted on the maximum rate of flux, Pi having no appreciable effect on the apparent affinity of the Na pump for either internal Na or external K. The effect of Pi is exerted along a rectangular hyperbola which tends to zero as Pi tends to infinity and is half-maximal at about 17 mM internal Pi. 2. Pi does not modify the rate of Na:Na exchange catalysed by the Na pump. 3. A reduction in the intracellular concentration of ATP reduces the maximum rate of Na:K exchange having no effect on the apparent affinity for either internal Na or external K. 4. The effects of ATP and Pi are mutually independent. 5 The lack of effect of ATP and Pi on the apparent affinity for internal Na is compatible with the idea that the affinity of the inner sites of the Na pump remains constant during a pump cycle. 6. The lack of effect of ATP on the apparent affinity for external K and the independence between the effects of ATP and Pi are difficult to explain if the only effect of ATP were its combination at a phosphorylating site. 7. The apparent affinities for K and phosphate become independent of the concentration of ATP, if it is assumed that in our experimental range the phosphorylating site is fully saturated with ATP, the rate of pumping being controlled by the state of occupation of a second non-phosphorylating site whose affinity for ATP is much lower. 8. The lack of effect of Pi on the apparent affinity for external K seems to indicate that during Na:K exchange the conformations of the pump that predominate are endowed with a reactivity towards inorganic phosphate and have the same high affinity for K in both their phospho and their dephospho states. 9. The kinetic behaviour of the Na pump in regard to its interactions with inner and outer cations, ATP and Pi seems to indicate that, in contrast with what happens with soluble allosteric proteins, in the active transport system ligand-induced changes in the reactivity are more important than ligand-induced changes in affinity. In this respect therefore the Na pump behaves as an allosteric 'V system'.

Adenosine Diphosphate↗