Charles DeLisi

The detection and classification of membrane-spanning proteins

Discriminant analysis can be used to precisely classify membrane proteins as integral or peripheral and to estimate the odds that the classification is correct. Specifically, using 102 membrane proteins from the National Biomedical Research Foundation (NBRF) database we find that discrimination between integral and peripheral membrane proteins can be achieved with 99% reliability. Hydrophobic segments of integral membrane proteins can also be distinguished from interior segments of globular soluble proteins with better than 95% reliability. We also propose a procedure for determining boundaries of membrane-spanning segments and apply it to several integral membrane proteins. For the limited data available (such as on transplantation antigens), the residues at the boundaries of a membrane-spanning segment are predictable to within the error inherent in the concept of boundary. As a specific indication of resolution, seven membrane-spanning segments of bacteriorhodopsin are resolved with no information other than sequence, and the predicted boundary residues agree with the experimental data on proteolytic cleavage sites. Several definitive but yet to be tested predictions are also made, and the relation to other predictive methods is briefly discussed. A computer program in FORTRAN for prediction of membrane-spanning segments is available from the authors.

Protein Antigenic Structures Recognized by T Cells; Potential Applications to Vaccine Design

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic structures. An algorithm to search for potential amphipathic helices from sequence information identified 18 of 23 known immunodominant T-cell epitopes from 12 proteins (p less than 0.001). Another statistical approach confirmed the importance of amphipathicity and also supported the importance of helical structure that had been proposed by others. It suggested that peptides able to form a stable secondary structure, especially a helix, more commonly formed immunodominant epitopes. We used this approach to predict potential immunodominant epitopes for induction of T-cell immunity in proteins of clinical relevance, such as the malarial circumsporozoite protein and the AIDS viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)

The biophysics of ligand-receptor interactions.

For the cells of an organism to act in the coordinated fashion necessary for complex functioning, they must be able to receive and transmit information. Information transfer is mediated by molecules released by the cells and may be local, as in the case of neurotransmitters, or long range, as in the case of hormones. It is apparent, however, that irrespective of the range of interaction, a cell must be able to distinguish, with a high degree of precision, the signals relevant to it from an enormous flow of background noise.Molecular recognition at the cell surface is mediated by receptors: cell surface glycoproteins that usually form an integral part of the plasma membrane (see, for example, Cuatrecasas & Greaves, 1978). Typically, receptors bind the ligands they are designed to recognize with affinities of the order of 108 M-1, and they translate that interaction into a sequence of signals that ultimately lead to biological activity.

Receptor clustering on a cell surface. I. theory of receptor cross-linking by ligands bearing two chemically identical functional groups

Abstract We present a general mathematical analysis of the problem of bivalent ligands, containing two identical reactive groups, interacting with cell surface receptors. The initial binding of such ligands to a cell is followed by further reactions on the surface, leading to the formation of ligand-receptor clusters. If the receptors are monovalent, at most two receptors can be cross-linked by ligand. However, if the receptors are bivalent (or multivalent), large cell surface aggregates can form, the clusters being distributed in size. Our mathematical analysis of ligand binding and receptor cross-linking is based upon the equivalent-site hypothesis and encompasses both equilibrium and kinetic aspects of the problem. In principle, the mathematical description of cluster formation kinetics requires the solution of an infinite system of coupled nonlinear differential equations. We show that in the absence of ligand-receptor rings, the problem of obtaining the entire aggregate size distribution as a function of time can be reduced to the solution of two nonlinear differential equations. Approximate solutions to these equations are developed via singular perturbation methods. When ring formation is included in the model, additional equations need to be solved. We show that when the rate constant for ring closure varies inversely with chain size, the infinite system of differential equations needed to describe the growth of linear aggregates can be reduced to a system of three integral and integrodifferential equations. To determine the concentration of rings, an additional equation must be solved for each ring size of interest. The mathematical development is general and may be applicable to the analysis of a number of biological problems, including histamine release from basophils and mast cells, B-cell triggering, and the action of hormones, such as insulin and epidermal growth factor.

Prediction of protein function from sequence properties: Discriminant analysis of a data base

The protein superfamilies in the National Biomedical Research Foundation sequence data base cluster into six groups that can be distinguished on the basis of four variables characterizing amino acid composition and local sequence properties. The variables are average hydrophobicity, net charge, sequence length and periodic variation in hydrophobic residues along the chain. The clusters they distinguish are: globins; chromosomal proteins; contractile system proteins and respiratory proteins other than cytochromes; enzyme inhibitors and toxins; enzymes except hydrolases; and all other proteins. The overall probability of correctly allocating a given protein to one of these functional groups is 0.76, with the allocation reliability being highest for globins (0.97) and for chromosomal proteins (0.93).

The effect of cell size and receptor density on ligand--receptor reaction rate constants.

Abstract A method is developed for calculating rate constants for ligands interacting with cell-bound receptors in terms of rate constants for reactions between solubilized molecules. The results indicate that for identical reaction mechanisms and typical cellular and molecular dimensions, the rate and equilibrium constants for reaction of solution phase ligand with receptors that are cell-bound or otherwise fixed, as on a bead, can be several orders of magnitude smaller or larger than their values for reactions with dispersed receptors. These variations have little to do with diffusion coefficient differences, but are intimately connected to the size and density of receptors, and the relation between diffusive and reactive rate constants. The main equations are tabularized and their utility illustrated by a brief discussion of data from the literature.

Immune surveillance and neoplasia—1 a minimal mathematical model

A deterministic predator-prey model is presented for describing the dynamics of a solid tumor in the presence of a specifically reactive lymphocyte population which is stimulated by, and antagonistic to, the tumor. The qualitative behavior of the solutions is developed and briefly compared to the results of transplantation experiments. Although the model is primitive, it leads to predictions that are in general agreement with observation and intuitive expectations. In particular, it is found that: (1) very low levels of transplanted tumor will not survive in the recipient. (2) At somewhat higher levels, tumor growth will be uncontrolled in the syngeneic recipient. However, immune intervention if early enough, can lead to control and elimination of the tumor. (3) At still higher levels of transplanted tumor, no amount of immune intervention will be effective in controlling the tumor. (4) If the recipients immune system is suppressed prior to transplantation, or is debilitated for any reason, the chance that the tumor will grow increases. (5) If the recipients immune system is stimulated prior to transplantation, the chance of tumor survival decreases. (6) The survival of the tumor is much more sensitive to changes in tumor parameters (for example, antigenicity) than in lymphocyte parameters. In addition it makes the unintuitive prediction that (7) There areisolated instances under which anincrease in the number of lymphocytes canincrease the chance of tumor survival.

Determination of equilibrium and rate constants by affinity chromatography

Abstract A theory of column chromatography is developed that includes the interaction of macromolecules with matrix-bound ligand and free ligand distributed throughout the column. The theory differs from previous formulations in its exact treatment of chemical kinetics and mass transfer kinetics. Characteristics of the elution profile — specifically the mean and variance— are expressed in terms of variables such as mobile phase velocity, bed height, ligand concentrations, mass transfer rate constants and chemical reaction rate constants. Equations are developed for monovalent and bivalent binding, for binding to ligands on porous and on impenetrable bed particles and for heterogeneous macromolecules. Previous results for the profile peak appearing in the literature are shown to be special cases of the equations presented here, holding only when the rate constants satisfy certain constraints. Our results therefore broaden the range of applicability of chromatography for the study of macromolecular interactions and precisely define its limits with respect to the determination of both thermodynamic and kinetic parameters.

Mean residence time—theoretical development, experimental determination, and practical use in tracer analysis☆

Abstract This paper examines the relationship between calculations of kinetic quantities from moments of tracer data with calculations of the same quantities based on the rate constants of a compartmental model describing the data. Most kinetic measures obtained from moments of the data are shown to be simple algebraic functions of compartmental mean residence times. New, relatively simple expressions for calculating the zeroth, first and higher order moments of the residence time of material in the system from the compartmental rate constants are developed. Under certain circumstances the moments of the data yield kinetic quantities different from those obtained using compartmental mean residence times. This analysis illustrates the additional kinetic insight that can be achieved through compartmental analysis.

A Theory of Measurement Error and Its Implications for Spatial and Temporal Gradient Sensing During Chemotaxis

In order that cells respond to environmental cues, they must be able to measure ambient ligand concentration. Concentrations fluctuate, however, because of thermal noise, and one can readily show that estimates based on concentration values at a particular moment will be subject to substantial error. Cells are therefore expected to average their estimates over somelimited time period. In this paper we assume that a cell uses fractional receptor occupancy as a measure of ambient ligand concentration and develop general expressions for the error a cell makes because the length of the averaging period is necessarily limited.Our analysis is general, relieving many of the assumptions underlying the seminal work of Berg and Purcell. The most important formal difference is our inclusion of occupancy-dependent dissociation—a phenomenon that has been well-documented for many systems. In addition, our formulation permits signal averaging to begin before chemical equilibrium has been established and it allows binding kinetics to be nonlinear (i.e., biomolecular rather than pseudo-first-order).The results are applied to spatial and temporal concentration gradients. In particular we estimate the minimum averaging times required for cells to detect such gradients under typical in vitro conditions. These estimates involve assigning numerical values to receptor ligand rate constants. If the rate constants are at their maximum possible values (limited only by center of mass diffusion), then either temporal or spatial gradients can be detected in minutes or less. If, however, as suggested by experiments, the rate constants are several orders of magnitude below their diffusion-limited values, then under typical constant gradient conditions the time required to detect a spatial gradient is prohibitively long, whereas temporal gradients can still be detected in reasonable lengths of time. This result was obtained for large cells such as lymphocytes, as well as for the smaller, bacterial cells. The ratio of averaging times for the two mechanisms—amounting to several orders of magnitude—is well beyond what could be reconciled by limitations of the calculation, and strongly suggests heavy reliance on temporal sensing mechanisms under typical in vitro conditions with constant spatial gradients.