cecropin-p1-li and maleimide

Molecular structures such as conformation and orientation are crucial in determining the activity of peptides immobilized to solid supports. In this study, sum frequency generation (SFG) vibrational spectroscopy was applied to investigate such structures of peptides immobilized on self-assembled monolayers (SAMs). Here cysteine-modified antimicrobial peptide cecropin P1 (CP1) was chemically immobilized onto SAM with a maleimide terminal group. Two important characteristics, length of the poly(ethylene glycol) (PEG) segment in the SAM and location of the cysteine residue in the peptide, were examined using SFG spectroscopy to determine the effect of each on surface immobilization as well as peptide secondary structure and its orientation in the immobilized state. Results have shown that while each length of PEG chain studied promotes chemical immobilization of the target peptide and prevents nonspecific adsorption, CP1 immobilized on long-chain (PEG2k) maleimide SAMs shows random coil structure in water, whereas CP1 demonstrates α-helical structure when immobilized on short-chain (with four ethylene glycol units - (EG4)) maleimide SAMs. Placement of the cysteine residue at the C-terminus promotes the formation of α-helical structure of CP1 with a single orientation when tethered to EG4 maleimide SAM surfaces. In contrast, immobilization via the N-terminal cysteine of CP1 results in a random coil or lying-down helical structure. The bacteria capturing/killing capability was tested, showing that the surface-immobilized CP1 molecules via C- and N- terminal cysteine exhibit only slight difference, even though they have different secondary structures and orientations.

Topics: Adsorption; Immobilized Proteins; Maleimides; Models, Molecular; Molecular Structure; Peptides; Protein Structure, Secondary; Surface Properties

Biosensors using peptides or proteins chemically immobilized on surfaces have many advantages such as better sensitivity, improved stability, and longer shelf life compared to those prepared using physically adsorbed biomolecules. Chemical immobilization can better control the interfacial conformation and orientation of peptides and proteins, leading to better activity of these biomolecules. In this research, molecular dynamics (MD) simulations were employed to systematically investigate the structure and dynamics of surface-tethered antimicrobial peptide cecropin P1 (CP1) modified with a cysteine residue at the C- (CP1c) or N-terminus (cCP1). Such CP1c and cCP1 molecules were chemically immobilized onto a silane-EG4-maleimide self-assembled monolayer (SAM) surface by forming a thio-ether bond between the cysteine group in CP1c or cCP1 and the surface maleimide group. The simulation results showed that the immobilized cCP1 (via the N-terminus) tends to bend and gradually lie down onto the SAM surface, due to the large structural fluctuation of the C-terminus induced by unfavorable interactions between the hydrophobic C-terminal residues and water. Differently, the tethered CP1c (via the C-terminus) more or less stands up on the surface, only tilting slightly even after 60 ns. The simulation results can be well correlated to the recent experimental results obtained from sum frequency generation (SFG) vibrational spectroscopic study. The current simulation data provide more atomic level details on how the hydrophobicity difference in the C-terminus and N-terminus of the amphiphilic peptide can lead to different structures of the same peptide tethered to the surface via different termini. This knowledge can be used to rationally design chemically immobilized peptides to achieve desired structure and functionality.

Topics: Amino Acid Sequence; Anti-Infective Agents; Cysteine; Immobilized Proteins; Maleimides; Molecular Dynamics Simulation; Peptides; Protein Structure, Secondary; Surface Properties; Vibration; Water

Sum frequency generation (SFG) vibrational spectroscopy has been applied to the investigation of peptide immobilization on a polymer surface as a function of time and peptide conformation. Surface immobilization of biological molecules is important in many applications such as biosensors, antimicrobial materials, biobased fuel cells, nanofabrication, and multifunctional materials. Using C-terminus-cysteine-modified cecropin P1 (CP1c) as a model, we investigated the time-dependent immobilization behavior in situ in real time. In addition, potassium phosphate buffer (PB) and mixtures of PB and trifluoroethanol were utilized to examine the effect of peptide secondary structure on CP1c immobilization to polystyrene maleimide (PS-MA). The orientation of immobilized CP1c on PS-MA was determined using polarized SFG spectra. It was found that the peptide solution concentration, solvent composition, and assembly state (monomer vs dimer) prior to immobilization all influence the orientation of CP1c on a PS-MA surface. The detailed relationship between the interfacial peptide orientation and these immobilization conditions is discussed.

Topics: Amino Acid Sequence; Buffers; Cysteine; Immobilized Proteins; Kinetics; Maleimides; Models, Molecular; Molecular Sequence Data; Peptides; Phosphates; Polystyrenes; Potassium Compounds; Protein Multimerization; Protein Structure, Quaternary; Reducing Agents; Solutions; Solvents; Surface Properties; Trifluoroethanol

A surface sensitive second order nonlinear optical technique, sum frequency generation vibrational spectroscopy, was applied to study peptide orientation on polymer surfaces, supplemented by a linear vibrational spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy. Using the antimicrobial peptide Cecropin P1 as a model system, we have quantitatively demonstrated that chemically immobilized peptides on polymers adopt a more ordered orientation than less tightly bound physically adsorbed peptides. These differences were also observed in different chemical environments, for example, air versus water. Although numerous studies have reported a direct correlation between the choice of immobilization method and the performance of an attached biological molecule, the lack of direct biomolecular structure and orientation data has made it difficult to elucidate the relationship between structure, orientation, and function at a surface. In this work, we directly studied the effect of chemical immobilization method on biomolecular orientation/ordering, an important step for future studies of biomolecular activity. The methods for orientation analysis described within are also of relevance to understanding biosensors, biocompatibility, marine-antifouling, membrane protein functions, and antimicrobial peptide activities.

Topics: Adsorption; Amino Acid Sequence; Biopolymers; Immobilized Proteins; Maleimides; Molecular Sequence Data; Peptides; Polystyrenes; Spectroscopy, Fourier Transform Infrared; Surface Properties; Vibration