Interactions of tryptophan, tryptophan peptides, and tryptophan alkyl esters at curved membrane interfaces

Abstract

Motivated by ongoing efforts to understand the mechanism of membrane protein crystallogenesis and transport in the lipidic cubic phase, the nature of the interaction between tryptophan and the bilayer/aqueous interface of the cubic phase has been investigated. The association was quantified by partitioning measurements that enabled the free energy of interaction to be determined. Temperature-dependent partitioning was used to parse the association free energy change into its enthalpic and entropic components. As has been observed with tryptophan derivatives interacting with glycerophospholipid bilayers in vesicles, tryptophan partitioning in the cubic phase is enthalpy driven. This is in contrast to partitioning into apolar solvents, which exhibits the classic hydrophobic effect whose hallmark is a favorable entropy change. These results with tryptophan are somewhat surprising given the simplicity, homogeneity, and curvature of the interface that prevails in the case of the cubic phase. Nevertheless, the interaction between tryptophan and the mesophase is very slight as revealed by its low partition coefficient. Additional evidence in support of the interaction was obtained by electronic absorption and fluorescence spectroscopy and fluorescence quenching. Partitioning proved insensitive to the lipid composition of the membrane, examined by doping with glycerophospholipids. However, the interaction could be manipulated in meaningful ways by the inclusion in the aqueous medium of salt, glycerol, or urea. The effects seen with tryptophan were amplified rationally when measurements were repeated using tryptophan alkyl esters and with tryptophan peptides of increasing length. These findings are interpreted in the context of the insertion, folding, and function of proteins in membranes.

Figure 1

Distribution of tryptophans in integral membrane proteins of known structure. The proteins included in the figure were selected from the ‘unique protein family’ subset in the Membrane Protein Data Bank (http://www.mpdb.ul.ie, Raman et al., 2005). The C_α-backbone trace of the protein is shown in grey (cyan in the case of 2C3E, panel B4) while the tryptophans are represented in black space-filling form. Proteins are arranged alphabetically by name, abbreviated in the interest of space, and by PDB accession number. The dotted lines shows the approximate location of the membrane/aqueous interfaces and are ∼30 Å apart. Positioning of proteins in the membrane is based on the original literature or a best guess by the authors. Models have been scaled to fit the panel. No particular vectoral orientation of proteins across the membrane has been imposed other than generally to place the bulkier extramembranal domain in the upper part of each panel. The view of the protein model shown was chosen to optimize visualizing the distribution of tryptophans in the vicinity of the interface. In the interest of space, only truly unique membrane protein structures are shown. Thus, for example, the photosynthetic reaction center from Blastochloris (Rps.) viridis is included in the figure while that from R. sphaeroides is not. The following proteins do not contain tryptophans and were omitted from the figure in the interest of space: ATP synthase (1QO1); glutamate transporter homologue (1XFH); large conductance mechanosensitive channel, MSCL (1MSL); outer membrane protein, NSPA (1P4T); sodium ATPase, F-type (1YCE); sodium ATPase, V-type (2BL2). In panel B4, a single cardiolipin molecule is shown in space-filling form with the following atom type coloring scheme: carbon, grey; oxygen, red; orange, phosphorous.

Figure 1

Distribution of tryptophans in integral membrane proteins of known structure. The proteins included in the figure were selected from the ‘unique protein family’ subset in the Membrane Protein Data Bank (http://www.mpdb.ul.ie, Raman et al., 2005). The C_α-backbone trace of the protein is shown in grey (cyan in the case of 2C3E, panel B4) while the tryptophans are represented in black space-filling form. Proteins are arranged alphabetically by name, abbreviated in the interest of space, and by PDB accession number. The dotted lines shows the approximate location of the membrane/aqueous interfaces and are ∼30 Å apart. Positioning of proteins in the membrane is based on the original literature or a best guess by the authors. Models have been scaled to fit the panel. No particular vectoral orientation of proteins across the membrane has been imposed other than generally to place the bulkier extramembranal domain in the upper part of each panel. The view of the protein model shown was chosen to optimize visualizing the distribution of tryptophans in the vicinity of the interface. In the interest of space, only truly unique membrane protein structures are shown. Thus, for example, the photosynthetic reaction center from Blastochloris (Rps.) viridis is included in the figure while that from R. sphaeroides is not. The following proteins do not contain tryptophans and were omitted from the figure in the interest of space: ATP synthase (1QO1); glutamate transporter homologue (1XFH); large conductance mechanosensitive channel, MSCL (1MSL); outer membrane protein, NSPA (1P4T); sodium ATPase, F-type (1YCE); sodium ATPase, V-type (2BL2). In panel B4, a single cardiolipin molecule is shown in space-filling form with the following atom type coloring scheme: carbon, grey; oxygen, red; orange, phosphorous.

Figure 2

Cartoon representation of cubic-Pn3m phase. Lipids are shown with the glycerol headgroup as an ellipse and the acyl chain as a wavy black line. Aqueous channels are shown in green and blue in the upper panel and in light blue in the lower panel. The phase is drawn to scale and represents that formed by monoolein at 40 %(w/w) water and 20 °C (16).

Figure 3

Fluorescence characteristics of tryptophan and its alkyl ester derivatives in SPP buffer and in cubic-Pn3m phase of hydrated monoolein at 20 °C. λ_max (A) and F_c, _max (B) are the wavelength and corrected intensity of maximum fluorescence emission. Data are shown as the average of at least three replicate sample preparation and fluorescence measurements. Error bars correspond to the standard deviation. Occasionally, the statistical error is smaller than the size of the symbol.

Figure 4

Fluorescence characteristics of tryptophan peptides in SPP buffer and in the cubic-Pn3m phase of hydrated monoolein at 20 °C. λ_max (A) and F_c, _max (B) are the wavelength and corrected intensity of maximum fluorescence emission. Data are shown as the average of at least three replicate sample preparation and fluorescence measurements. Error bars correspond to the standard deviation. Occasionally, the statistical error is smaller than the size of the symbol.

Figure 5

Accessibility of tryptophan in SPP buffer and in the cubic-Pn3m phase of monoolein at 20 °C to the water-soluble quencher, bromide, as judged by fluorescence quenching. Bromide concentration refers to the potassium bromide concentration in the water channels of the cubic phase. Fluorescence data have been corrected for background fluorescence from buffer and lipid and for the inner filter effect, and have been normalized to the quencher-free (F_c,0) value. Data are shown as the average of at least three replicate sample preparations and fluorescence measurements. Error bars correspond to the standard deviation. Occasionally, the statistical error is smaller than the size of the symbol. Stern-Volmer analysis (Bolen and Holloway, (25)) has been applied to the quenching of tryptophan in SPP buffer. The corresponding plot of F_c,0/F_c versus bromide concentration is linear in the range examined with a correlation coefficient of 0.99 and a Stern-Volmer quenching constant of 0.80 M⁻¹. An analysis of quenching behaviour in the more complex cubic phase system was not undertaken in this study (see also legend to Figure 6).

Figure 6

Fluorescence quenching of tryptophan and its alkyl (A) and peptide derivatives (B) in the cubic-Pn3m phase of hydrated monoolein at 20 °C. Bromo-MAG is the quenching lipid and its concentration is expressed as mole% corresponding to 100 (mole bromo-MAG) / [(mole bromo-MAG) + (mole monoolein)]. Flourescence data have been corrected for background fluorescence from SPP buffer and lipid and for the inner filter effect, and have been normalized to the quencher-free (F_c,0) value. Data are shown as the average of at least three replicate sample preparations and fluorescence measurements. Error bars correspond to the standard deviation. Lines (double exponentials) have been drawn to guide the eye. Given the complex nature of the system under investigation where fluorophors are distributed to varying degrees between two very different environments, the bilayer interface and the aqueous solution, a more complete analysis was not considered informative at this stage and was not undertaken.

Figure 7

Release profiles of tryptophan peptides from the cubic-Pn3m phase of hydrated monoolein as a function of time at 20 °C. The solid lines represent fits to the release data based on average D_F and k values obtained from the diffusion model (Table S6). Error bars corresponding to the standard deviation are included for all data points and are based on triplicate measurements.

Figure 8

Temperature-dependence of the partitioning of tryptophan and its derivatives into the cubic-Pn3m phase of hydrated monoolein. The partition coefficient, expressed in mole fraction units, is shown as a function of peptide size at 20 °C (A), alkyl chain length at 20 °C (B) and temperature (C). The free energy of partitioning is shown in (D) as a function of temperature and was calculated using Eq. 2 and the data in (C). Data are shown as the average of at least three replicate sample preparations and partitioning measurements. Error bars correspond to the standard deviation. In some cases (panels A and B) the statistical error is smaller that the size of the symbols.

Figure 8

Temperature-dependence of the partitioning of tryptophan and its derivatives into the cubic-Pn3m phase of hydrated monoolein. The partition coefficient, expressed in mole fraction units, is shown as a function of peptide size at 20 °C (A), alkyl chain length at 20 °C (B) and temperature (C). The free energy of partitioning is shown in (D) as a function of temperature and was calculated using Eq. 2 and the data in (C). Data are shown as the average of at least three replicate sample preparations and partitioning measurements. Error bars correspond to the standard deviation. In some cases (panels A and B) the statistical error is smaller that the size of the symbols.