Two collagen-mimetic peptides, CP+ and CP-, are reported in which the sequences comprise a multiblock architecture having positively charged N-terminal (Pro-Arg-Gly)(3) and negatively charged C-terminal (Glu-Hyp-Gly)(3) triad extensions, respectively. CP+ rapidly self-associates into positively charged nanosheets based on a monolayer structure. In contrast, CP- self-assembles to form negatively charged monolayer nanosheets at a much slower rate, which can be accelerated in the presence of calcium(II) ion. A 2:1 mixture of unassociated CP- peptide with preformed CP+ nanosheets generates structurally defined triple-layer nanosheets in which two CP monolayers have formed on the identical surfaces of the CP+ nanosheet template. Experimental data from electrostatic force microscopy (EFM) image analysis, zeta potential measurements, and charged nanoparticle binding assays support a negative surface charge state for the triple-layer nanosheets, which is the reverse of the positive surface charge state observed for the CP+ monolayer nanosheets. The electrostatic complementarity between the CP+ and CP- triple helical cohesive ends at the layer interfaces promotes a (CP-/CP+/CP-) compositional gradient along the z-direction of the nanosheet. This structurally informed approach represents an attractive strategy for the fabrication of two-dimensional nanostructures with compositional control.