Jonathan S. Wall¹, Fayad M. Ayoub², and Paul S. O'Shea

¹ Human Immunology & Cancer Program, University of Tennessee Medical Center at Knoxville, 1924 Alcoa Highway, Knoxville, TN 37920-6999. USA.

² Department of Biological & Chemical Sciences, University of Essex, Colchester, Essex, England U.K.

Received 01/11/96; Accepted 05/29/96; On-line 08/16/96

It has previously been demonstrated that the fluorescent probe, FPE, may be stably incorporated into biological and synthetic membranes, allowing monitoring of the binding of proteins (34, 35, 36). After the initial protein-membrane contact has taken place it is then possible to characterise any protein reorientation which may follow. In order to investigate the potential physiologic effect of Bence-Jones proteins, the interaction of lambdaRG57 with cell membranes was studied. The binding of lambdaRG57 to lymphocyte and synthetic membranes was shown to be comprised of two distinct events. The first event presumably represents a collision limited binding process followed by a slower reorganisation event. The secondary event is believed to be associated with the maximisation of coulombic attractive forces between the protein and the membranes, as well as entropically (and/or enthalpically) driven interaction as a result of the hydrophobic effect. The technique of using FPE as a probe for monitoring changes in s facilitates the real-time visualization of the proteins charged moieties when they come into close proximity to the membrane surface (Fig. 3). Once the protein-membrane contact has been made, FPE may report any protein reorganisation events which are accompanied by a modification of the charge profile present at the membrane-water interface (34, 35, 36).

The lack of interaction shown in the KSCN media, together with the parity of the results obtained in the other experimental media suggest that the interaction of lambdaRG57 with the PLV membrane is governed predominantly by electrodynamic forces, rather than Coulombic attraction. In simpler terms, the hydrophobic effect, is thought to be the major force directing the binding of lambdaRG57 to the PLV membrane.

This conclusion is reached on the basis that in a medium where the major anion is SCN^- little binding is observed. It is believed that the high entropy of hydration of SCN^- (49) disrupts the non-polar properties of the phospholipid bilayer thereby modifying/removing the lambdaRG57 binding site. This ultimately results in the observed loss of binding of the protein in this medium (Fig. 2 & Table 1).

The observation that an addition of lambdaRG57 results in a decrease in the fluorescence intensity of FPE suggests that Psi_s is becoming more electronegative. It has been shown however, that the net charge carried by lambdaRG57 at this pH is positive (Fig. 1). These observations may be reconciled in light of the mode of action of FPE (34), which is only responsive to charged species residing within the immediate vicinity of the lipid-water interface, as dictated by the Debye length. However, if the charged groups move away from the mebrane surface into the bulk, or become buried within the lipid phase of the membrane, as a result of binding, a reversal of the apparent net charge may occur (34). If this interpretation is valid it would be expected that the interaction of lambdaRG57 with the membrane surface involves more than just a simple surface-binding event. The multi-component interaction model for lambdaRG57, is given support by the results of the stopped-flow experiments, discussed below (Fig. 3).

The far-UV CD spectra of lambdaRG57, under all experimental conditions tested, are characterised by a single (negative) minimum at approximately 218nm and a positive band at 200nm. This is indicative of an anti-parallel ß-sheet configuration (see e.g., 50). IgLCs are composed of two rigid anti-parallel, ß-sheet "boxes", each constrained by a disulphide bridge (20). It may be assumed that any movement of the protein once bound must involve a global reorientation of the whole protein complex. After binding, circular dichroic studies indicated no change in the secondary structure of lambdaRG57, and the protein remained in its predominantly ß-sheet configuration, as shown by the single minimum at 218nm, in both high and low ionic strength media, as well as in the absence or presence of liposomes (Fig. 9).

The effect of temperature on the fast phase of the lambdaRG57-membrane association (Fig. 4 and Table 3), believed to be the binding event, suggests that there are two effects which may be responsible for this process. The first, a slow event with a temperature insensitive rate constant of 1.5 mol^-1sec^-1, combined with a rapid event which increases dramatically over the temperature ranges studied. It is interesting to postulate that these two components represent the electrostatic and hydrophobic components respectively. This is in accordance with the data for lambdaRG57 binding in 100mM KSCN (Fig. 2) which indicates that the major factor determining the association of the protein with a lipid membrane is a hydrophobic interaction. Furthermore, the estimated activation energy which corresponds to the temperature sensitive, kinetic phase is 13.53 KJmol^-1. This is indicative of an interaction which requires less energy than that for purely ionic interactions.

The slower, reorientation phase of the lambdaRG57 interaction with PLVs, takes place over 50 seconds. (Fig. 3B). This event is accurately described by a single exponential decay, the rate of which increases from 7.1 x 10^-4 sec^-1 at 2.8°C to 12.2 sec^-1 at 38.3°C (Table 3). The activation energy associated with this process is calculated to be 87.78 KJmol^-1 (Fig. 8 & Table 3). This molecular process therefore, appears to require 6.5 times more energy than the binding process, which is consistent with the view that the reorganisation of large domains of the protein is occurring.

These data show very clearly a rapid increase in fluorescence intensity over the first 500 milli-seconds, which is believed to be the binding process which results in Psi_s becoming increasingly electropositive. This phase is then followed by a sustained decrease in fluorescence intensity, or an increase in the electronegativity of Psi_s, which extends over a period of more than 50 seconds.

The binding of IgLCs to synthetic and B-cell membranes appears to involve a complex series of events, rather than simple adherence due to electrostatic attraction. It seems likely therefore, that the biological effects of these proteins are linked to many inherent factors, and not just to the pI of the protein, a relationship which has been previously criticised (51). Indeed, it has been seen that there is little correlation between the net charge of an IgLC and the efficiency of binding to simple, model membranes, as shown in this study. If any attempts are to be made to limit the binding of IgLCs to normal cells in a clinical situation, or to prevent high order aggregates forming and becoming deposited systemically, then it appears more likely that this will be achieved by the abrogation of the hydrophobic forces which are believed to govern these processes.

The reorientation of the IgLC upon its binding to the membrane surface may facilitate its interaction with integral mebrane proteins. Such interactions may lead to the initiation of transmembrane signals via membrane protein aggregation. Alternatively, the membrane-bound IgLC may adopt a configuration which initiates, or enhances the formation of light chain fibrils, associated with amyloidosis. The role of the membrane surface in the aetiology of amyloidosis is unclear but its' involvement is undergoing investigation (unpublished data). Furthermore, the reorientation event induced by the IgLC-membrane surface interaction, raises the question as to whether other proteins use non-specific binding mechanisms as the first step of interaction with cell membranes. This may also be a means to re-configure their structure in such a way as to facilitate a specific ligand-receptor interaction or transmembrane insertion and thereby possibly mediate a cellular response.