Crystallographic Characterization of N-Oxide Tripod Amphiphiles

Abstract

Tripod amphiphiles are designed to promote the solubilization and stabilization of intrinsic membrane proteins in aqueous solution; facilitation of crystallization is a long-range goal. Membrane proteins are subjects of extensive interest because of their critical biological roles, but proteins of this type can be difficult to study because of their low solubility in water. The nonionic detergents that are typically used to achieve solubility can have the unintended effect of causing protein denaturation. Tripod amphiphiles differ from conventional detergents in that the lipophilic segment contains a branchpoint, and previous work has shown that this unusual amphiphilic architecture can be advantageous relative to traditional detergent structures. Here we report the crystal structures of several tripod amphiphiles that contain an N-oxide hydrophilic group. The data suggest that tripods can adapt themselves to a nonpolar surface by altering the hydrophobic appendage that projects toward that surface and their overall orientation relative to that surface. Although it is not possible to draw firm conclusions regarding amphiphile association in solution from crystallographic data, trends observed among the packing patterns reported here suggest design strategies to be implemented in future studies.

Hydrophobic variants of ganglio-tripod amphiphiles for membrane protein manipulation

“…At this time, it is not clear whether or not ganglio-TPAs form small PDCs relative to those formed with DDM. However, our previous study may shed light on the topic [37]. We recently reported the crystal structures of several N -oxide TPAs and found that these amphiphiles form thinner nonpolar layers than do conventional detergents in their crystalline lattice [37].…”

“…However, our previous study may shed light on the topic [37]. We recently reported the crystal structures of several N -oxide TPAs and found that these amphiphiles form thinner nonpolar layers than do conventional detergents in their crystalline lattice [37]. The means by which these TPAs arrange themselves therein show how the hydrophobic surface displayed by one TPA layer can accommodate that of an adjacent TPA layer.…”

“…The development of tripod amphiphiles (TPAs) as membrane mimetics has worked to coordinate the chemical and physical properties of these molecules and their associated micelles to ensure utility in membrane protein research [33–37]. Since one cannot a priori predict these properties, they need to be tested empirically.…”

“…This carbon in the lipophilic region limits the conformational freedom of this class of molecules, thereby making them rigid relative to conventional detergents. This rigidification likely enabled us to solve the crystal structures of several N -oxide TPAs themselves [37] and has the potential to facilitate the crystallization of a wide array of membrane proteins; TPA-solubilized bR and potassium channel from Streptomyces lividans have been crystallized, although their structures have not yet been solved [34,36]. Recent TPA advances had led to a series of molecules with bifurcated glucose headgroups with favorable solubilization and stabilization efficacy [35].…”

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

“…To test for detergents compatible with GOAT octanoylation, we screened initially at 1.5 × critical micelle concentration and at 1 mM for the following detergents: Anzergent 3–14, APO 10, Big Chap, Brij35, C13E8, CHAPS, CHAPSO, Cholate, CycloFos 6, CycloFos 7, Cymal 6, Cymal 7, Cy-TripGlu[28], Deoxy Big Chap, Deoxycholate, Digitonin, Dodecyl Maltoside, Fos-Choline-12, Fos-Choline-13, Fos-Choline-16, GDN [29], GNG-3 (Glucose Neopentyl Glycol)[30], Hexadecyl Maltoside, LysoFos-Choline 14, LysoFos-Choline 18, MNG-3 (Maltose Neopentyl Glycol)[31], Octyl Glucoside, Ph-TripGlu[28], Sucrose Monododecanoate, Taurocholate, TDAO (Tetradecyl Dimethylamine Oxide), Tetradecyl Dimethyl Glycine, Tetradecyl Maltoside, TRIPAO[32], and Triton X-100. Mixed micelles of cholesteryl hemisuccinate (CHS) plus CHAPS, MNG-3 (Lauryl Maltose Neopentyl Glycol), or Fos-Choline-16 were made by mixing detergent:CHS at ratios of 5:1, 10:1, and 20:1 (w/w), respectively, and added analogously at varying concentrations.…”