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H M McConnell

Publications and source records attributed to H M McConnell.

At least 19 recordsLinked to original sources
PubMed1999

Conformational isomers of a class II MHC-peptide complex in solution.

A number of kinetic measurements of peptide dissociation from class II MHC-peptide complexes provide compelling evidence for the existence of conformational isomers in solution. There is evidence that T-lymphocytes can distinguish such isomers. However, virtually nothing is known about the structure of these isomers. Accordingly, we have investigated a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c residues 89-104 (PCC) by 19F-NMR. Two fluorine labels were placed on the PCC peptide; one fluorine label was placed at a MHC contact site, the other at a position involved in T-cell receptor (TCR) recognition. Introduction of these labels did not alter the observed kinetics of the PCC/I-Ek complex. The NMR data show two conformational isomers of this immunogenic complex. The presence of conformational isomers at a TCR contact site suggests that these structures may be recognized differently by the TCR. The agreement between the dissociation kinetics and the 19F-NMR data demonstrate that kinetic heterogeneity is correlated with structural counterparts observed by NMR. Dissociations in the presence of dimethyl sulfoxide were used to show that the rate of interconversion of these conformational isomers at pH 7.0 is low, with a lifetime on the order of hours or more. Modification of a peptide residue of PCC occupying the minor MHC binding pocket P6 alters the 19F-NMR spectra of both labels. This demonstrates that distant changes of amino acid residues can influence the conformation of the whole antigenic peptide inside the MHC binding cleft.

Animals↗
PubMed1998

Kinetic isomers of a class II MHC-peptide complex.

Class II major histocompatibility (MHC) molecules bind fragments of antigens and present them to T cells. The triggering of the T-cell receptor (TCR) of CD4(+) T-helper cells by these protein-peptide complexes is a key event in the generation of a cellular immune response. In the context of this interaction, it is generally assumed that class II MHC-peptide complexes adopt a single recognition structure at the cell surface. On the other hand, kinetic analysis has revealed that a number of class II MHC-peptide complexes show biphasic dissociation kinetics, indicating the presence of multiple kinetic isomers. Here, we demonstrate that a water-soluble version of the murine class II MHC molecule I-Ek complexed with an antigenic peptide derived from pigeon cytochrome c (PCC) displays monophasic as well as biphasic dissociation kinetics. While a simple monophasic dissociation curve was obtained at neutral pH, the complex showed biphasic dissociation behavior at acidic pH. This shift was independent of the ionic strength of the solution. Moreover, the short-lived isomer could be regenerated from a pool of kinetically homogeneous long-lived complexes. This demonstrates that the isomers interconvert and exist in a pH-sensitive equilibrium. Altering the peptide residue of PCC that occupies the P6 pocket of I-Ek results in a class II MHC-peptide complex that shows only monophasic dissociation, indicating that the glutamine at this position plays a key role in the kinetic heterogeneity of the complex.

Amino Acid Sequence↗
PubMed1998

Evidence that the autoimmune antigen myelin basic protein (MBP) Ac1-9 binds towards one end of the major histocompatibility complex (MHC) cleft.

The NH2-terminal peptide of myelin basic protein (MBP) bound to the class II major histocompatibility complex (MHC) protein I-Au is an immunodominant epitope in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. However, the MBP-I-Au complex is very unstable. To investigate this, we performed site-directed mutagenesis of the I-Au MHC protein and the MBP peptide. Biochemical, T cell activation, and molecular modeling studies of mutant complexes demonstrate that the MBP peptide's key residue for MHC binding, lysine 4, is buried in the P6 pocket of I-Au, which is predominantly hydrophobic. This implies that the MBP-I-Au complex differs from more stable complexes in two respects: (a) the peptide leaves the NH2-terminal region of the MHC peptide-binding cleft unoccupied; (b) the peptide is not anchored by typical favorable interactions between peptide side chains and MHC pockets. To test these hypotheses, a modified MBP peptide was designed based on molecular modeling, with the aim of producing strong I-Au binding. Extension of the NH2 terminus of MBP with six amino acids from the ova peptide, and replacement of the lysine side chain in the P6 pocket with an aromatic anchor, results in >1,000-fold increased binding stability. These results provide an explanation for the unusual peptide-MHC-binding kinetics of MBP, and should facilitate an understanding of why mice are not tolerant to this self-peptide- MHC complex.

Amino Acid Sequence↗
PubMed1998

Electric field-induced critical demixing in lipid bilayer membranes.

Electric fields can induce lateral reorganization of lipids in fluid bilayer membranes. The resulting concentration profiles readily are observed in planar-supported bilayers by epifluorescence microscopy. When a fluorescently labeled lipid was used to probe the field-induced separation of cardiolipin from egg-phosphatidylcholine, an enhanced sensitivity to the electric field was observed that is attributed to a critical demixing effect. A thermodynamic model of the system was used to analyze the results. The observed concentration profiles can be understood if the lipid mixture has a critical temperature equal to 75 degrees K. The steady-state distribution of lipids under the influence of an electric field is very sensitive to demixing effects, even at temperatures well above the critical temperature for spontaneous phase separation, and this may have significant consequences for organization and structural changes in natural cell membranes.

Cardiolipins↗
PubMed1998

Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected].

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Amino Acid Sequence↗
PubMed1998

Formation of a highly peptide-receptive state of class II MHC.

Peptide binding to class II MHC proteins occurs in acidic endosomal compartments following dissociation of class II-associated invariant chain peptide (CLIP). Based on peptide binding both to empty class II MHC and to molecules preloaded with peptides including CLIP, we find evidence for two isomeric forms of empty MHC. One (inactive) does not bind peptide. The other (active) binds peptide rapidly, with k(on) 1000-fold faster than previous estimates. The active isomer can be formed either by slow isomerization of the inactive molecule or by dissociation of a preformed peptide/MHC complex. In the absence of peptide, the active isomer is unstable, rapidly converting to the inactive isomer. These results demonstrate that fast peptide binding is an inherent property of one isomer of empty class II MHC. Dissociation of peptides such as CLIP yields this transient, peptide-receptive isomer.

Amino Acid Sequence↗
PubMed1997

Electric field-induced reorganization of two-component supported bilayer membranes.

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

Journal Article↗
PubMed1997

Liquid-liquid immiscibility in lipid monolayers.

Some binary lipid mixtures form coexisting liquid phases when spread at the air/water interface. This work describes the pressure-composition phase diagrams of binary mixtures of four unsaturated phosphatidylcholines with dihydrocholesterol. These four binary mixtures have critical compositions of approximately fifty mole percent, and average critical exponents of 0.25 +/- 0.07. The data can also be approximated by a regular solution thermodynamic model, yielding parameters for the non-ideality of these mixtures.

Cholestanol↗
PubMed1997

Specific T cell recognition of kinetic isomers in the binding of peptide to class II major histocompatibility complex.

Helper T cells are triggered by molecular complexes of antigenic peptides and class II proteins of the major histocompatibility complex. The formation of stable complexes between class II major histocompatibility complex proteins and antigenic peptides is often accompanied by the formation of a short-lived complex. In this report, we describe T cell recognition of two distinct complexes, one short-lived and the other long-lived, formed during the binding of an altered myelin basic protein peptide to I-Ak. One myelin basic protein-specific T cell clone is triggered by only the short-lived complex, and another is triggered by only the stable complex. Thus, a single peptide bound to a particular class II molecule can activate different T cells depending on the conditions of the binding reaction.

Animals↗
PubMed1997

Kinetics and extent of T cell activation as measured with the calcium signal.

We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

Amino Acid Sequence↗
PubMed1997

Screening for novel drug effects with a microphysiometer: a potent effect of clofilium unrelated to potassium channel blockade.

Changes in cellular metabolism in response to pharmacological compounds can be detected using a biosensor known as a microphysiometer, which measures the rate at which cells release acidic metabolites. We have applied this technique to screen for effects of cation channel blockers on the metabolism of a variety of human and murine cell lines. At concentrations sufficient for cation channel blockade, most of these drugs have little or no effect on cellular metabolism as measured by acid release. In contrast, the potassium channel blocker clofilium triggers sustained increases in acid release at low (> or = 3 microM) concentration. Acid release persists in media containing high (150 mM) extracellular potassium. This release is not triggered by chemically similar potassium channel blockers. Thus these metabolic effects reflect a potent and specific function of clofilium which is unrelated to potassium channel blockade. Attempts to identify physiological correlates to this response revealed that low concentrations of clofilium but not other potassium channel blockers cause lymphoma apoptosis. These findings demonstrate that effects of clofilium found in other studies may not be due to changes in plasma membrane potassium conductance.

Adrenergic beta-Antagonists↗
PubMed1996

Equilibration rates in lipid monolayers.

A theoretical analysis is given for the rate of change of domain sizes in lipid monolayers at the air-water interface. The calculation is applicable to liquid domains formed from binary mixtures of lipids that form two coexisting liquid phases. Under conditions where the two lipid molecules have approximately equal areas, the equilibration rate does not involve macroscopic hydrodynamic flow in the subphase but rather depends on the diffusion coefficient of the lipid molecules. The calculation shows that the equilibration rate in binary mixtures of cholesterol and phosphatidylcholine is remarkably slow, the radius of a typical 20-microns diameter domain changing by as little as a part in a million per second. Under these circumstances, equilibration times of the order of days of weeks are expected. Even with such long times, the final state reached by the monolayer will in general be a state of metastable equilibrium, rather than true equilibrium.

Cholesterol↗
PubMed1996

Molecular modeling and design of invariant chain peptides with altered dissociation kinetics from class II MHC.

We have used molecular modeling to design substitutions in an invariant chain-derived peptide (CLIP), so as to alter the stability of its complex with class II major histocompatibility complex (MHC) proteins. We sought first to test whether CLIP binds in the same way to different class II MHC proteins. We designed destabilizing substitutions of two residues (Met 91 and Met 99) previously predicted to act as the major anchor residues for binding to all class II MHC and measured their effect on CLIP's dissociation rate from a series of three murine I-A MHC proteins. Even a conservative substitution preserving size and hydrophobicity but reducing flexibility (leucine, a branched residue) caused large accelerations in dissociation rates (up to 25-fold) at either position in all three MHC alleles, supporting the consistent role of these positions as the major anchors for MHC binding. These data also support the view that the special flexibility of the methionine side chains at these positions is essential for binding to diverse MHC molecules. We also used molecular modeling to design allele-specific enhancements of peptide binding. Designed substitutions of CLIP Pro 96 by Ala (for Ad), Glu (Ak), and Tyr (Au) each yielded strong enhancement of binding (up to 128-fold) for their targeted allele and only moderate or destabilizing effects to the other alleles. These results demonstrate the accuracy of the molecular models and the predictive value of this modeling. Moreover, they provide strong evidence for the proposed general model of invariant chain association, indicating that it binds to all class II MHC in the same conformation.

Alleles↗
PubMed1996

Early biochemical signals arise from low affinity TCR-ligand reactions at the cell-cell interface.

The kinetics of acid release by a mixture of T cells and antigen presenting cells were measured with a microphysiometer during a brief exposure to antigenic peptides. We find that some of the early biochemical events that lead to cellular proliferation cause a specific increase in the rate of acid release. The duration of this increase in acid release reflects the life-time of the peptide-MHC complexes. Peptides that form long-lived complexes produce a response that is stable for more than an hour. Serial TCR engagement is suggested by the observation that the amplitude of this stable response can be rapidly shifted up or down with additional agonist peptide or with antibodies that block T cell receptor binding. Cells briefly exposed to a peptide that forms short-lived peptide-MHC complexes produce a response that decays rapidly as peptide is washed away. A quantitative analysis of the kinetics of this decay in acidification demonstrates that intercellular TCR-ligand reactions are rapid, reversible, and of low apparent affinity with < 20% of peptide-MHC ligand bound to a TCR at any one time. These results demonstrate that the fraction of peptide-MHC ligands bound to TCRs at the cell-cell interface is no higher than anticipated from the affinities observed in solution for isolated TCRs and ligands.

Amino Acid Sequence↗
PubMed1996

Critical pressures in multicomponent lipid monolayers.

Epifluorescence microscopy has been used previously to study coexisting liquid phases in lipid monolayers of dihydrocholesterol and dimyristoylphosphatidylcholine at the air/water interface. This binary mixture has a critical point at room temperature (22 degrees C), a monolayer pressure of approx. 10 mN/m, and a composition in the vicinity of 20-30 mol% dihydrocholesterol. It is reported here that this critical pressure can be lowered, raised, or maintained constant by systematically replacing molecules of this phosphatidylcholine with molecules of a phosphatidylethanolamine, or an unsaturated phosphatidylcholine, or mixtures of the two, while maintaining the dihydrocholesterol concentration at 20 mol%. Thus, even complex mixtures of lipids may be characterized by a single, well-defined second-order phase transition. In principle, such transitions might be found in biological membranes.

Cholestanol↗
PubMed1996

Kinetic discrimination in T-cell activation.

We propose a quantitative model for T-cell activation in which the rate of dissociation of ligand from T-cell receptors determines the agonist and antagonist properties of the ligand. The ligands are molecular complexes between antigenic peptides and proteins of the major histocompatibility complex on the surfaces of antigen-presenting cells. Binding of ligand to receptor triggers a series of biochemical reactions in the T cell. If the ligand dissociates after these reactions are complete, the T cell receives a positive activation signal. However, dissociation of ligand after completion of the first reaction but prior to generation of the final products results in partial T-cell activation, which acts to suppress a positive response. Such a negative signal is brought about by T-cell ligands containing the variants of antigenic peptides referred to as T-cell receptor antagonists. Results of recent experiments with altered peptide ligands compare favorably with T-cell responses predicted by this model.

Animals↗