Table 1.
Data-collection and scattering-derived parameters.
Figure 1.
(A) Experimental scattering curves (o) with error bars in gray color and (insert) the Guinier plot with the linear fit (red line) are shown. (B) The fitting curves (—; green: fit for the experimental data, red: fit for the calculated ab initio model) of HAMLET derived from SAXS data. (C) Distance distribution functions of HAMLET. (D) The Kratky plot indicates that the protein is globular and folded. The increase at higher angles might reflect that the protein is slightly flexible.
Figure 2.
SAXS structure of HAMLET and native human α-lactalbumin crystal structure.
(A) Low resolution structures of HAMLET in brown spheres and (B) its 180° view. (C–D) Superposition of HAMLET with human α-lactalbumin (PDB id: 1B9O [21]). The C-terminal residues from L105 to L123 of the crystal structure of the human α-lactalbumin, which form a flexible loop in the crystal structure of human α-lactalbumin, are colored red. We suggest that this region L105 to L123 takes up an extended conformation in HAMLET by forming a tail.
Figure 3.
(A) Amino acid sequence and secondary structure of human α-lactalbumin as well as the peptide domains alpha1, alpha2 and beta. (B) The three peptide domains in the crystallographic structure of human α-lactalbumin (PDB id: 1B9O [21]) (C), which is superimposed into the solution shape of HAMLET (C). (D) Surface representation showing the low resolution structure of the human α-lactalbumin with the three domains; alpha 1 in red, alpha 2 in blue and beta in gray color. The tail region of the SAXS shape, which is modeled on to the alpha2 domain low resolution structure, is also shown in blue.
Figure 4.
Internalization of peptides into tumor cells and changes in tumor cell morphology.
(A) Internalization of peptides. A549 lung carcinoma cells cultured on glass slides, were incubated with peptide-oleate mixtures for 1 hour, fixed and stained with AlexaFluor568-streptavidin, counterstained with WGA and examined by confocal microscopy. Alpha1 and alpha2 peptides, mixed with oleate, were internalized as shown by the red fluorescence. The beta peptide was not internalized. Scale bar 20 µm. (B) Morphological changes in A549 lung carcinoma cells treated with HAMLET, alpha1 peptide+oleate, alpha2 peptide+oleate and beta peptide+oleate recorded by holography imaging. Cells treated with HAMLET started to round up after 30 minutes and after 60 minutes, many cells had detached. Alpha1 peptide+oleate mixture triggers similar morphological changes as that by HAMLET. Alpha2 peptide+oleate mixture triggers similar morphological changes. Beta peptide+oleate mixture did not change cell morphology.
Figure 5.
Ion fluxes and tumor cell death.
(A) Peptides trigger ion fluxes in tumor cells. The free intracellular concentration of Na+, K+ and Ca2+ were measured by fluorescence spectrometry using CoroNa Green, FluxOR and Fluo-4, respectively. HAMLET, alpha1 peptide+oleate and alpha2 peptide+oleate mixtures trigger rapid fluxes of all three ions. Intracellular potassium ion concentrations were reduced due to ion efflux, while those of sodium and calcium were increased. Mean of at least two experiments. P values are explained in the text. (B) Peptide-oleate mixtures kill tumor cells. A549 lung carcinoma cells and Jurkat leukemia cells were incubated with HAMLET, oleate or peptide-oleate mixtures for 3 hours. Cell death was quantified as ATP levels and PrestoBlue, in % of control.
Figure 6.
Internalization of small peptides and induction of ion fluxes.
(A) Internalization of biotinylated peptides (red) by A549 lung carcinoma cells counterstained with WGA (green) and Hoechst (blue) and examined by confocal microscopy. Peptides 1,10 and 11 were internalized in the absence of oleate and peptides 10 and 11 also in the presence of oleate. (B) K+ and (C) Ca2+fluxes in tumor cells triggered by petides 1, 10 and 11 were measured by fluorescence spectrometry. Peptides 10 and 11 triggered Ca2+ fluxes, in the presence and absence of oleate and peptide 6 a weaker Ca2+ flux with oleate. (D) Insignificant Na+ fluxes.