Final Report Summary - PROADS (Molecular Recognition: Understanding Proteins Adsorption to Inorganic Surfaces)
In nature, composite materials with unique properties such as high mechanical strength, optical functionality or electronic structure are formed through the specific interactions between organic (usually proteins) and inorganic materials. For example, the combination of collagen and hydroxyapatite leads to the formation of bones which serve as a mechanical and supportive tissue. Like proteins, short peptides also exhibit specific binding affinity towards inorganic surfaces. In nanotechnology, the specificity of these binders (mainly peptides) is used in various applications, particularly in the design of new biomimetic hybrid materials. Moreover, the specific binding affinity of peptides towards inorganic substrates has been utilized to prepare nanostructured materials with novel properties and functions, such surface biocompatibility, drug delivery, crystal growth regulation, and nanoparticle synthesis. In addition, understanding this binding mechanism will allow us to design effective biomedical materials having applications in bone-based research including bone and dentin tissue engineering, tendon and ligament repair, and enamel formation. Many approaches including phage display and Surface Plasmon Resonance (SPR) have been employed to identify the affinity of peptides towards targeted inorganic surfaces. Although, these methods indeed spot many peptide sequences that bind to certain surfaces, the reason for the propensity of a certain sequence to a certain substrate is still not clear. This is because completely different sequences can bind a specific substrate. Single molecule force spectroscopy using Atomic Force Microscopy (AFM) may provide information about peptide-inorganic surface interaction at single molecular level. Force spectroscopy allows the detailed study of molecular interactions that are not available with other techniques. This technique has been extensively used to investigate the interaction between biotin and–avidin, antigen–antibody, peptide-cell interaction etc.
During this project we established a single molecule force spectroscopy by AFM system to examine the interactions between organic materials such as amino acids and peptides with inorganic materials. Using this system we measured the interactions of individual amino acids with inorganic substrates in aqueous solution. In each measurement, an amino acid residue (lysine, glutamate, phenylalanine, leucine, or glutamine) each represents a class of amino acids (positively or negatively charged, aromatic, nonpolar, and polar) was connected to the AFM tip. Force–distance curves measured the interaction of the individual amino acid bound to the AFM tip with a silicon substrate or mica. Using this method, we were able to measure low adhesion forces and could clearly determine the strength of interactions between the individual amino acid residues and the inorganic substrate. In addition, we observed how changes in the pH and ionic strength of the solution affected the adsorption of the residues to the substrates (Langmuir 2013). Our results pinpointed the important role of hydrophobic interactions among the amino acids and the substrate, where hydrophobic phenylalanine exhibited the strongest adhesion to a silicon substrate. (Figure 1) Additionally, electrostatic interactions also contributed to the adsorption of amino acid residues to inorganic substrates. A change in the pH or ionic strength values of the buffer altered the strength of interactions among the amino acids and the substrate.(Figure 2) We concluded that the interplay between the hydrophobic forces and electrostatic interactions will determine the strength of adsorption among the amino acids and the surface. Overall, these results contribute to our understanding of the interaction at the organic–inorganic interface. These results may have implications for our perception of the specificity of peptide binding to inorganic surfaces.
Furthermore, using this system and computer simulations (with Prof. Carlos Alemán, Madrid) we studied the binding of peptides towards inorganic substrates. By performing alanine scan we examined the propensity of each amino acid in a seven amino acid peptide to bind the substrate (mica). Our results indicate that the binding of the peptide to the substrate is not controlled by the specific sequence of the peptide, but rather by the conformational freedom of the peptide in solution versus its freedom on the substrate. When the conformational freedom of the peptide in solution is identical to its freedom on the substrate the peptide will not adhere to the substrate. However, when the conformational freedom is reduced on the surface the binding will occur. These results are of great significance for the design of new composite materials (publication in preparation).
Our next steps include further study of the interactions of peptides to inorganic materials. In addition, we will also exploit this AFM force spectroscopy system to study the interaction between DNA sequences and transcription factors.
During this project we established a single molecule force spectroscopy by AFM system to examine the interactions between organic materials such as amino acids and peptides with inorganic materials. Using this system we measured the interactions of individual amino acids with inorganic substrates in aqueous solution. In each measurement, an amino acid residue (lysine, glutamate, phenylalanine, leucine, or glutamine) each represents a class of amino acids (positively or negatively charged, aromatic, nonpolar, and polar) was connected to the AFM tip. Force–distance curves measured the interaction of the individual amino acid bound to the AFM tip with a silicon substrate or mica. Using this method, we were able to measure low adhesion forces and could clearly determine the strength of interactions between the individual amino acid residues and the inorganic substrate. In addition, we observed how changes in the pH and ionic strength of the solution affected the adsorption of the residues to the substrates (Langmuir 2013). Our results pinpointed the important role of hydrophobic interactions among the amino acids and the substrate, where hydrophobic phenylalanine exhibited the strongest adhesion to a silicon substrate. (Figure 1) Additionally, electrostatic interactions also contributed to the adsorption of amino acid residues to inorganic substrates. A change in the pH or ionic strength values of the buffer altered the strength of interactions among the amino acids and the substrate.(Figure 2) We concluded that the interplay between the hydrophobic forces and electrostatic interactions will determine the strength of adsorption among the amino acids and the surface. Overall, these results contribute to our understanding of the interaction at the organic–inorganic interface. These results may have implications for our perception of the specificity of peptide binding to inorganic surfaces.
Furthermore, using this system and computer simulations (with Prof. Carlos Alemán, Madrid) we studied the binding of peptides towards inorganic substrates. By performing alanine scan we examined the propensity of each amino acid in a seven amino acid peptide to bind the substrate (mica). Our results indicate that the binding of the peptide to the substrate is not controlled by the specific sequence of the peptide, but rather by the conformational freedom of the peptide in solution versus its freedom on the substrate. When the conformational freedom of the peptide in solution is identical to its freedom on the substrate the peptide will not adhere to the substrate. However, when the conformational freedom is reduced on the surface the binding will occur. These results are of great significance for the design of new composite materials (publication in preparation).
Our next steps include further study of the interactions of peptides to inorganic materials. In addition, we will also exploit this AFM force spectroscopy system to study the interaction between DNA sequences and transcription factors.