J. Phys. Chem. B 2000, 104, 7431-7438
7431
Protein Deformation and Surfactancy at an Interface Stephen A. Holt,† Duncan J. McGillivray,† Simon Poon,‡ and John W. White*,† Research School of Chemistry, Australian National UniVersity, Canberra, ACT 0200 Australia, and CooperatiVe Research Centre for Bioproducts, School of Botany, UniVersity of Melbourne, ParkVille, VIC 3010, Australia ReceiVed: February 17, 2000; In Final Form: May 18, 2000
The deformation of a protein, myoglobin and two derivatives, apomyoglobin and peptide (1-55) has been studied at the air/water interface. The filled monolayer has been shown to have a thickness of ca. 16 Å for the three materials, despite the different tertiary structures involved. It is proposed that the tertiary structure of the myoglobin and apomyoglobin is modified at the interface and it is the R-helical secondary structure which is the major determinant of the observed layer thickness. Additionally a small effect of the heme cofactor on the surface structure is identified for myoglobin not for apomyoglobin. The unfolding of the tertiary structure of myoglobin was greatest at the isoelectric point, coinciding with the largest surface excess. This unfolding seems to occur in the surface layer only, the partially filled second layer which forms at higher concentrations is about the same size as the solution protein dimensions.
Introduction Proteins and their fragments have interesting amphipathic properties arising from the hydrophilic and hydrophobic qualities of the constituent amino acids and from the secondary and tertiary structure generated by folding in solution. The availability of neutron and X-ray reflectometry techniques allows protein structure at the air/water interface to be studied at nanometer resolution and recent experiments1-4 have shown that for globular proteins such as β-lactoglobulin the molecular form is remarkably resilient to adsorption. In this paper we report studies on myoglobin and a short peptide fragment from it which is an active emulsifying agent. Our aim is to use information from the study of structure at the air/water interface to infer the behavior of the peptide at the oil/water interface and to model the way in which the protein conformation alters upon adsorption. In considering the response of a globular protein to adsorption at an interface, it is worth recalling that the molecule, as crystallized, contains approximately 74% water and is greatly segmented. It can therefore be expected that time dependent phenomena in the adsorption will be observed, related first to the diffusion-limited adsorption onto the surface from the bulk, which may occur in a time scale of minutes. It has been shown that the surface tension of a myoglobin solutions5,6 with a freshly formed interface decreases to a steady value over about 30 min. As a second step, possibly on a much longer time scale, there may be relaxation processes associated with change in the protein’s conformation and shape. Such relaxation processes may involve anything from a simple change in the shape of the molecule to complete denaturation. We are thus concerned here to present data on the equilibrium structures which arise after relaxation is complete. We study the pH dependence and make some preliminary observations on the rate processes associated with the equilibration. We analyze the factors controlling this behavior in terms of the * To whom correspondence is to be addressed. E-mail: John.White@ anu.edu.au. Fax: +61 2 6249 4903. † Australian National University. ‡ University of Melbourne.
expected rigidity of the structure (related possibly to the number of disulfide bonds), secondary structure, such as alpha helices and beta sheets, and the distribution of hydrophilic, hydrophobic and charged regions in the molecule, which may be linked to the solution pH. Myoglobin has been chosen for this work because it is a well understood “model” protein with well studied solution and crystal structures. It has a well-defined tertiary structure, consisting of eight alpha-helices, many of which are amphipathic, arranged in a folding pattern known as the “globin fold”. This fold encases the functional heme group in myoglobin and is stabilized by the nonbacked interactions between the group and the protein. Furthermore, in the crystal, the helices are aligned such that there are stabilizing packing arrangements in which the ridges formed by the side chains of one set of helices fit into the grooves of adjacent helices. Myoglobin is far more susceptible to extensive conformational change when adsorbed at an interface than the more rigid globular proteins such as lysozyme,7 primarily due to the absence of disulfide linkages found in the cysteine residues of the “rigid” proteins. Myoglobin is stable in aqueous solution down to pH 4 and is readily available. The molecule is also an interesting test case as it affords the possibility of studying the effect of the heme cofactor on its surface structure, achieved through the indirect route of comparing the results from the holo- and the apo-protein. Additionally our work also includes the study of the myoglobin derivative, peptide (1-55). Compositionally the only difference between myoglobin and apomyoglobin is the lack of the heme cofactor in the latter. There has been some debate as to the role the heme group plays in maintaining the stability of the protein, and to whether the structure of the apoprotein is the same as that of the holoprotein (i.e., metmyoglobin).8-11 There are over 200 published myoglobin crystal structures12 and none for apomyoglobin, indicating that the cofactor may affect the availability of crystalline samples of the apoprotein. This implies that the apoprotein may not be as “structured” as myoglobin, which in turn points to some heme group influence on the structure. This conjecture is reinforced
10.1021/jp000614q CCC: $19.00 © 2000 American Chemical Society Published on Web 07/18/2000
7432 J. Phys. Chem. B, Vol. 104, No. 31, 2000
Holt et al. TABLE 1: Listing of the Protein Concentrations Studieda
Figure 1. Structure of peptide (1-55), inferred from the crystal structure of myoglobin.
by the high binding affinity of the heme group for the protein, K ) 3 × 1014,11 which is indicative of extremely strong stabilizing interactions between the side chains of the protein chain and the heme group. Solution measurements have however shown that apomyoglobin does tend to adopt a similar geometry to that of myoglobin.13 Poon et al.5 cleaved apomyoglobin into three peptides, with the fragment containing amino acids (1-55) found to be the most effective with regards to the emulsification of oil-in-water mixtures. As can be seen in Figure 1 this fragment consists of essentially two major helices with a small portion of disordered chain. This structural assignment requires the assumption that the fragment maintains the same secondary structure as the equivalent section of the protein, since a crystal structure of the fragment has not been determined. As such, peptide (155) gives an opportunity to study the extent to which secondary structure has a role to play in the surface properties of the protein, without the complicating effects of the protein’s intricate tertiary folding. This material is also a potentially useful industrial surfactant. Experimental Section The myoglobin used was horse skeletal muscle myoglobin, obtained from Sigma-Aldrich (catalog no. M0630) at greater than 95% purity, used as delivered. The apomyoglobin was produced from horse heart myoglobin, Sigma-Aldrich (catalog no. M1882), using a method5 based on that in refs 14 and 15 to remove the heme group. The apomyoglobin produced contained less than 1% heme by UV-vis analysis. This was then cleaved using cyanogen bromide in formic acid, to yield after separation the peptide (1-55), which consists of the first 55 residues of myoglobin. Each of these materials was kept below 0 °C when pure and below 4 °C when made into solution. Each solution was kept for a maximum of 1 week once made up. The protein solutions were made using Milli-Q Millipore water, resistivity of 18 MΩ cm or greater. The surface tension of the Milli-Q water varied by no more than (0.2 mN m-1 at 20 °C over 24 h. A series of buffered solutions were also made up using a 50 mM H2PO4-/HPO42- system weighed out from solids for pH 7, and buffers at other pH values by CRC recipes.16 X-ray and neutron reflectivity experiments were carried out at the Research School of Chemistry, Australian National University, and the ISIS facility, Rutherford Appleton Laboratory, respectively. We present the specular reflectivity data as a function of the scattering vector Qz (Qz ) 4π/λ sin θ where λ is the wavelength and θ the incident and exit angle of the beam to the surface). Consequently Qz can be varied, by altering either the incident angle or the wavelength of the radiation. The X-ray reflectometer17 uses the former method operating at a fixed wavelength with the scattering vector varied by simultaneously altering the incident and detection angles. The neutron
concn (mg/mL)
myoglobin
0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1.0
X X X X X X X
apomyoglobin
peptide (1-55)
X X X X X X
X X X* X* X* X* X X
a At least one X-ray and one neutron (D2O subphase) data set were collected for each concentration. * indicates that data on an ACMW subphase was also collected.
reflectometer, SURF,18 operates in an energy dispersive mode, where Qz is varied by introducing a beam with range of wavelengths to the surface at a fixed angle. The alignment of the reflectometer was tested by measuring the reflectivity of a sample of Millipore water for X-rays and D2O for neutrons, and comparing the observed model parameters with the wellestablished values available. A background data set, acquired by offsetting the exit arm of the X-ray reflectometer by 0.5°, has been subtracted from all X-ray measurements. The background setting is well away from any specular reflection and it is preferable to measure the background for angular dispersive instruments as the volume of material sampled changes as a function of angle and a flat background is not sufficient. This is in contrast to the neutron experiments where the instrument has a fixed geometry and the dominant background is due to incoherent scattering, which was accounted for in the modeling process by subtraction of a flat background extrapolated from the scattering at high Qz. The reflectivity experiments can be divided into two groups, first concentration dependent and second studies at fixed concentration with varying pH. In addition it is possible to use mixtures of D2O and H2O to produce a solution with the same scattering power as air for neutrons. This Air Contrast Matched Water (ACMW) was used in a subset of the neutron experiments to confine the scattering contrast to the protein material. The range of concentrations was produced for each of the samples through serial dilution from a standard solution, made up from sample weighed out and dissolved into pure water in a volumetric flask. Table 1 lists the experiments performed as a function of concentration. Myoglobin at 0.1 mg/mL was studied as a function of pH from 6 to 11 in unit steps. Samples were introduced into a clean thermostated Teflon trough and up to 1 h was allowed for temperature equilibration (as the measurements were performed at 25 °C and the samples stored at
