Racemic protein crystallography has been proven to

Racemic protein crystallography has been proven to be a powerful technique for crystallizing proteins , , , , . The structures of many difficult-to-crystallize peptides/proteins of different sizes were resolved by the racemic protein crystallography combined with the protein chemical synthesis , , , , , . In this work, we report the chemical synthesis of natural linaclotide (-type) and its enantiomeric -peptide (-type) by using Fmoc solid phase peptide synthesis (Fmoc-SPPS) method. The X-ray crystal structure of linaclotide was determined in 1.59Å by racemic protein crystallography. The crystal structure showed that linaclotide has a tight, three-beta turns spatial structure immobilized by three pairs of disulfide bonds. Linaclotide is a heat-stable enterotoxin analog of consisting of 14 3902 mg with three pairs of intramolecular disulfide bonds (C1-C6, C2-C10 and C5-C13) (a) . The use of racemic crystallography for the crystallization of linaclotide needs to obtain -type and -type proteins. To this end, we used Wang resin as a solid scaffold and DIC (-diisopropylcarbodiimide) as condensing reagent to synthesize -type and -type linaclotide directly through Fmoc-SPPS . After the completion of the synthesis, the crude linaclotide was cleaved from the resin and the side chain protecting group was removed by using a cleaving reagent (TFA/HO/phenol/TIPS=87.5/5/5/2.5, v/v/v/v) (b) , , , , , , , , , , , , , , , . The -type and -type crude peptides were analyzed by analytical HPLC and verified by ESI-MS. As show in c/d, both crude peptides had good purity. After purification by RP-HPLC and lyophilized, we obtained -type and -type reduced linaclotide (isolated yield: 44.9% for -reduced linaclotide and 51.6% for -reduced linaclotide). We then folded the - and -type reduced linaclotide by using a redox pair consisting of oxidized glutathione (GSSH) and reduced glutathione (GSH). Both - and -type reduced linaclotide were first dissolved in 50mmol/L tris buffer, respectively, then GSSH and GSH were added to each buffer (molar ratio: peptide/GSSH/GSH=1/10/100). The pH of the buffer was adjusted to 8.0 and folding process was performed overnight at 25°C . As shown in a/b, the - and -type reduced linaclotide were almost completely converted to the corresponding - and -type linaclotide. After folding, the pH of the folded solution was adjusted to 2.0, then purified by HPLC immediately to give - and -type linaclotide (isolated yield: 51.7% for -linaclotide and 59.2% for -linaclotide). With - and -type linaclotide in hands, we first verified the difference in secondary structure between -type and -type linaclotide by circular dichroism (CD) spectra. As show in a, the CD spectrum of -type linaclotides indicates a negative and a positive peak in the range of 205–215nm and 195–205nm, respectively, while the curve of -linaclotide is symmetrically opposite to the -linaclotide. SDS-PAGE analysis (b) shows that -type and -type linaclotide appeared between 17 and 26kDa with a diffuse appearance. These results imply that the structure of linaclotide was sufficiently rigid, which could not be completely denatured under SDS conditions. After confirming the protein characterization, we carried out the racemic crystallization experiment to obtain the X-ray structure of linaclotide. Crystals were screened using a Hampton crystallization kit in 18°C crystal chamber after mix the - and -type linaclotide to their respective final concentrations of 10mg/mL. After three days, we obtained the racemic crystals of linaclotide. The crystals were grown in 0.2mol/L potassium sodium tartrate tetrahydrate, 20% (w/v) polyethylene glycol 3,350 to form a P1 space group with diffraction resolution of 1.59Å. Structural information of linaclotide was obtained after molecular replacement and refinement. As shown in a and b, the crystals of - and -type linaclotide pack together and are almost mirror-image linked together. Linaclotide stabilizes its spatial structure by three pairs of intramolecular disulfide bonds, giving it a compact spatial structure containing three-beta turns. Linaclotide has a similar amino acid sequence to heat-stable enterotoxins, which is a class of diarrhea-causing toxins secreted by intestinal pathogens. Through the structural comparison between linaclotide and heat-stable enterotoxin of (ST mimic), we conclude that linaclotide has similar spatial arrangement (c) , . The acquisition of linaclotide crystals potentially provides the basis for the subsequent optimization of the activity.