Tracking Peptide Molecules

As in various processes of immunology membrane proteins play a crucial role during HIV infection of the T-cell. One of the initial steps is the fusion of the viral membrane with the target T-cell membrane, a process catalyzed by a transmembrane domain of the HIV glycoprotein gp41.

Single-particle tracking techniques were developed to study transmembrane domains of the T-cell receptor and the fusion peptide of the gp41 protein. For this purpose both model peptides and peptides mimicking the transmembrane domains of the T-cell receptor and the fusion peptide of gp41 protein were studied using different model membranes and outer conditions.

Our major goal is to understand the kinetics and thermodynamics of the binding of peptides to the membrane and of their mutual interaction, as a function of membrane composition and structure.

Binding of a HIV-Virus involves the insertion of the fusion peptide into the target T-Cell. The fusion peptide interacts not only with the binding receptor CD4 but also with the T-Cell receptor complex (TCR) leading to an inhibition of its immunoactive function (Cohen, T., Biochem., 2008, 47, 4826-48).

 

 
 
 

Single Particle Tracking

Single-particle tracking (SPT) is a powerful tool of the fluorescence spectroscopy to study dynamics of heterogeneous systems such as the cell membranes. Each particle is thereby located with sub-diffraction resolution which allows analyzing of its individual dynamics instead of using the ensemble average.
The fluorescence characteristics of each single particle and its trace through space and time reveal information about its environment and possibly interactions. We use this technique to study interactions of single molecules within a model membrane.

For the detection each individual molecule is labelled with a fluorescent probe and excited by a laser. An inverted microscope with TIRF excitation creates a planar excitation volume which is ideal for the investigation of membranes (see figure SETUP). The movements of the fluorescent spots within the membrane are recorded by an ultra-sensitive and ultra-fast EM-CCD camera. The traces of the particles are later extracted and analyzed to find possibly interactions with other fluorescent or non-fluorescent particles.

Peptide-membrane interactions:
The insertion of a peptide and its motion within the membrane depend strongly on its nature: hydrophobic or hydrophilic. The 2D diffusive motion of the non-charged fusion peptide (FP) and doubly-charged T-cell receptor core peptide (CP) were studied using fluorescent labels. We varied the membrane temperature in order to test the effect of altering its fluidic properties on diffusion.

JUMP: Distance histogram of FP (blue) and CP (green) after 600 ms.

TEMPERATURES: Mobile fractions (MF) and mean diffusion coefficients at different temperatures of rhodamine-labeled single lipids (red) and FP (blue). The transition temperature between gel and liquid-crystal phase is 15°C.

→The fusion peptide diffuses independently of membrane conditions!

Peptide-peptide interactions:
The interaction between the CP and the FP was studied by adding fluorescently labeled CP together with different concentrations of unlabeled FP to the membrane.

INTENSITIES: Mean intensity per trace of the CP (green) and of FP-CP-complexes (blue).

CP-FP: Mobile fractions (MF) of the CP (green) and of the FP-CP-complex (blue) at different concentrations of FP.

→ Complexes likely include more than one core peptide.
→ Complex formation leads to increased mobility!

Conclusion & Outlook:

1. The hydrophobic nature of the FP leads to much faster diffusion than that of the CP.
2. Nevertheless, the diffusion of the FP is not influenced by changes of the fluidic properties of the membrane: It stays constant following phase transition even as the mobility of the phospholipids changes dramatically.
3. Although both peptides have a mobile fraction of about 40%, their complex is much more mobile (85%), while the diffusion coefficient stays comparable to that of a single CP.
4. The intensities of the formed complexes indicate that the majority consist of several peptides. Complex formation itself is independent of the FP concentration.

Future research will address the puzzles raised above:

• the relation between peptide diffusion and membrane phase.
• the stoichiometry of FP-CP complexes, complex formation dynamics and
• differences in complex-membrane interactions with respect to the individual peptides.