A Miniature Protein Stabilized by a Cation−π Interaction Network

The design of folded miniature proteins is predicated on establishing noncovalent interactions that direct the self-assembly of discrete thermostable tertiary structures. In this work, we describe how a network of cation−π interactions present in proteins containing “WSXWS motifs” can be emulated to stabilize the core of a miniature protein. This 19-residue protein sequence recapitulates a set of interdigitated arginine and tryptophan residues that stabilize a distinctive β-strand:loop:PPII-helix topology. Validation of the compact fold determined by NMR was carried out by mutagenesis of the cation−π network and by comparison to the corresponding disulfide-bridged structure. These results support the involvement of a coordinated set of cation−π interactions that stabilize the tertiary structure.

Timothy W. Craven, Min-Kyu Cho, Nathaniel J. Traaseth, Richard Bonneau, and Kent Kirshenbaum. (2016), A Miniature Protein Stabilized by a Cation−π Interaction Network. J. Am. Chem. Soc. 138(5), 1543-1550 Link

Antimicrobial Peptoid Membrane Pore-Forming Activity


Antimicrobial peptides (AMPs) are critical components of the innate immune system and exhibit bactericidal activity against a broad spectrum of bacteria. We investigated the use of N-substituted glycine peptoid oligomers as AMP mimics with potent antimicrobial activity. The antimicrobial mechanism of action varies among different AMPs, but many of these peptides can penetrate bacterial cell membranes, causing cell lysis. We previously hypothesized that amphiphilic cyclic peptoids may act through a similar pore formation mechanism against methicillin-resistant Staphylococcus aureus (MRSA). Peptoid-induced membrane disruption is observed by scanning electron microscopy and results in a loss of membrane integrity. We demonstrate that the antimicrobial activity of the peptoids is attenuated with the addition of polyethylene glycol osmoprotectants, signifying protection from a loss of osmotic balance. This decrease in antimicrobial activity is more significant with larger osmoprotectants, indicating that peptoids form pores with initial diameters of ∼2.0–3.8 nm. The initial membrane pores formed by cyclic peptoid hexamers are comparable in diameter to those formed by larger and structurally distinct AMPs. After 24 h, the membrane pores expand to >200 nm in diameter. Together, these results indicate that cyclic peptoids exhibit a mechanism of action that includes effects manifested at the cell membrane of MRSA.

Smith, P. T., Huang, M. L., Kirshenbaum, K. (2015), Osmoprotective polymer additives attenuate the membrane pore-forming activity of antimicrobial peptoids. Biopolymers, 103: 227–236. Link

BONLAC Technique for de Novo Protein Synthesis


Measuring the synthesis of new proteins in the context of a much greater number of pre-existing proteins can be difficult. To overcome this obstacle, bioorthogonal noncanonical amino acid tagging (BONCAT) can be combined with stable isotope labeling by amino acid in cell culture (SILAC) for comparative proteomic analysis of de novo protein synthesis (BONLAC). In the present study, we show that alkyne resin-based isolation of l-azidohomoalanine (AHA)-labeled proteins using azide/alkyne cycloaddition minimizes contamination from pre-existing proteins. Using this approach, we isolated and identified 7414 BONCAT-labeled proteins. The nascent proteome isolated by BONCAT was very similar to the steady-state proteome, although transcription factors were highly enriched by BONCAT. About 30% of the methionine residues were replaced by AHA in our BONCAT samples, which allowed for identification of methionine-containing peptides. There was no bias against low-methionine proteins by BONCAT at the proteome level. When we applied the BONLAC approach to screen for brain-derived neurotrophic factor (BDNF)-induced protein synthesis, 53 proteins were found to be significantly changed 2 h after BDNF stimulation. Our study demonstrated that the newly synthesized proteome, even after a short period of stimulation, can be efficiently isolated by BONCAT and analyzed to a depth that is similar to that of the steady-state proteome.

Zhang, G., Bowling, H.,  Hom, N., Kirshenbaum, K., Klann, E., Chao, M. V., Neubert, T. A. (2014), In-Depth Quantitative Proteomic Analysis of de Novo Protein Synthesis Induced by Brain-Derived Neurotrophic Factor. J Proteome Res., 13(12):5707-14. Link

Metal-Binding Peptoids and X-Ray Fluorescence


N-Substituted glycine peptoid oligomers have recently attracted attention for their metal binding capabilities. Due to their efficient synthesis on solid phase, peptoids are well suited for generation of compound libraries, followed by screening for molecular recognition and other diverse functional attributes. Ideally, peptoids could be simultaneously screened for binding to a number of metal species. Here, we demonstrate the use of bench-top X-ray fluorescence (XRF) instrumentation to screen rapidly, on solid support, a library of peptoid oligomers incorporating metal-binding functionalities. A subset of the peptoid sequences exhibited significant metal binding capabilities, including a peptoid pentamer and a nonamer that were shown to selectively bind nickel. The binding capabilities were validated by colorimetric assay and by depletion of Ni2+ ion concentration from solution, establishing bench-top XRF as a rapid, practicable high-throughput screening technique for peptoid oligomers. This protocol will facilitate discovery of metallopeptoids with unique material properties.

Nalband, D. M., Warner, B. P., Zahler, N. H., Kirshenbaum, K. (2014), Rapid identification of metal-binding peptoid oligomers by on-resin X-ray fluorescence screening. Biopolymers Peptide Science, 102: 407–415. Link