title: A genetic approach for controlling the binding and orientation of proteins on nanoparticles.
abstract: Although silver nanoparticles are excellent surface enhancers for Raman spectroscopy, their use to probe the conformation of large proteins at interfaces has been complicated by the fact that many polypeptides adsorb weakly or with a random orientation to colloidal silver. To address these limitations, we sought to increase binding affinity and control protein orientation by fusing a silver-binding dodecapeptide termed Ag4 to the C-terminus of maltose-binding protein (MBP), a well-characterized model protein with little intrinsic silver binding affinity. Quartz crystal microbalance measurements conducted with the MBP-Ag4 fusion protein revealed that its affinity for silver (Kd approximately 180 nM) was at least 1 order of magnitude higher than a control protein, MBP2, containing a non-silver-specific C-terminal extension. Under our experimental conditions, MBP-Ag4 SERS spectra exhibited 2-4 fold higher signal-to-background relative to MPB2 and contained a number of amino acid-assigned vibrational modes that were either weak or absent in control experiments performed with MBP2. Changes in amino acid-assigned peaks before and after MBP-Ag4 bound maltose were used to assess protein orientation on the surface of silver nanoparticles. The genetic route described here may prove useful to study the orientation of other proteins on a variety of SERS-active surfaces, to improve biosensors performance, and to control functional nanobiomaterials assembly.
authors.affiliation: Department of Chemical Engineering, University of Washington, Seattle, Washington 98195-1750, USA.
authors.forename: Atanu
authors.initials: A
authors.lastname: Sengupta
authors.forename: Corrine K
authors.initials: CK
authors.lastname: Thai
authors.forename: M S R
authors.initials: MS
authors.lastname: Sastry
authors.forename: James F
authors.initials: JF
authors.lastname: Matthaei
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Schwartz
authors.forename: E James
authors.initials: EJ
authors.lastname: Davis
authors.forename: François
authors.initials: F
authors.lastname: Baneyx
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D003460:Crystallization; D058619:Maltose-Binding Proteins; D053768:Metal Nanoparticles / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011791:Quartz; D012834:Silver / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
chemical_list: D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011506:Proteins; D011791:Quartz; D012834:Silver