Limits...
Structural genomics of membrane proteins.

Walian P, Cross TA, Jap BK - Genome Biol. (2004)

Bottom Line: Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts.There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts. There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

Show MeSH
Primary detergents used to obtain crystals of membrane proteins suitable for high-resolution structural studies [1]. The proportion of proteins solved with each detergent is indicated. Abbreviations: OG, n-octyl-β-D-glucopyranoside; NG, n-nonyl-β-D-glucopyranoside; OM, n-octyl-β-D-maltopyranoside; DM, n-decyl-β-D-maltopyranoside; UDM, n-undecyl-β-D-maltopyranoside; DDM, n-dodecyl-β-D-maltopyranoside; TDM, n-tridecyl-β-D-maltopyranoside; C8E4, polyoxyethylene (4) octyl ether; C12E8, polyoxyethylene (8) dodecyl ether; C12E9, polyoxyethylene (9) dodecyl ether; C10DAO, n-decyl-N,N-dimethylamine-N-oxide; LDAO, n-dodecyl-N,N-dimethylamine-N-oxide; LAPAO, 3-laurylamido-N,N-dimethylpropylaminoxide; FC14, n-tetradecylphosphocholine; MEGA10, decanoyl-N-methylglucamide; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC395774&req=5

Figure 2: Primary detergents used to obtain crystals of membrane proteins suitable for high-resolution structural studies [1]. The proportion of proteins solved with each detergent is indicated. Abbreviations: OG, n-octyl-β-D-glucopyranoside; NG, n-nonyl-β-D-glucopyranoside; OM, n-octyl-β-D-maltopyranoside; DM, n-decyl-β-D-maltopyranoside; UDM, n-undecyl-β-D-maltopyranoside; DDM, n-dodecyl-β-D-maltopyranoside; TDM, n-tridecyl-β-D-maltopyranoside; C8E4, polyoxyethylene (4) octyl ether; C12E8, polyoxyethylene (8) dodecyl ether; C12E9, polyoxyethylene (9) dodecyl ether; C10DAO, n-decyl-N,N-dimethylamine-N-oxide; LDAO, n-dodecyl-N,N-dimethylamine-N-oxide; LAPAO, 3-laurylamido-N,N-dimethylpropylaminoxide; FC14, n-tetradecylphosphocholine; MEGA10, decanoyl-N-methylglucamide; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine.

Mentions: As mentioned above, a detergent that is well-suited for protein solubilization may not be the detergent of choice for crystallization, and detergents can be exchanged through dialysis or during purification while the target protein is bound to a chromatographic column. Just as with the screening of detergents for the optimization of solubilization, a variety of detergents should be screened during the course of crystallization trials. An impressive range of detergents has been used to obtain crystals yielding high-resolution diffraction (Figure 2). From crystal-packing considerations, detergents with the potential to yield the smallest possible micelle region on the solubilized protein should best support the formation of the protein-to-protein contacts needed for crystallization. A potential downside is that smaller detergents tend to have higher CMCs, requiring higher concentrations to maintain protein solubility, and are more likely to destabilize native structure. The goal should therefore be to identify the smallest detergents that maintain homogeneous and monodisperse solutions of structurally sound target proteins. Alternatively, the use of secondary detergents or amphiphiles as additives to alter the properties of mixed micelles has also yielded high-quality crystals [27,28]. The location, or mere presence, of an affinity tag may play a role in determining whether a protein will crystallize, and it may be advisable to conduct crystallization trials on target proteins in which the polyhistidine affinity tags have been removed. Cleavage sites engineered into the expressed protein can be used to remove these tags using a detergent-resistant protease.


Structural genomics of membrane proteins.

Walian P, Cross TA, Jap BK - Genome Biol. (2004)

Primary detergents used to obtain crystals of membrane proteins suitable for high-resolution structural studies [1]. The proportion of proteins solved with each detergent is indicated. Abbreviations: OG, n-octyl-β-D-glucopyranoside; NG, n-nonyl-β-D-glucopyranoside; OM, n-octyl-β-D-maltopyranoside; DM, n-decyl-β-D-maltopyranoside; UDM, n-undecyl-β-D-maltopyranoside; DDM, n-dodecyl-β-D-maltopyranoside; TDM, n-tridecyl-β-D-maltopyranoside; C8E4, polyoxyethylene (4) octyl ether; C12E8, polyoxyethylene (8) dodecyl ether; C12E9, polyoxyethylene (9) dodecyl ether; C10DAO, n-decyl-N,N-dimethylamine-N-oxide; LDAO, n-dodecyl-N,N-dimethylamine-N-oxide; LAPAO, 3-laurylamido-N,N-dimethylpropylaminoxide; FC14, n-tetradecylphosphocholine; MEGA10, decanoyl-N-methylglucamide; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC395774&req=5

Figure 2: Primary detergents used to obtain crystals of membrane proteins suitable for high-resolution structural studies [1]. The proportion of proteins solved with each detergent is indicated. Abbreviations: OG, n-octyl-β-D-glucopyranoside; NG, n-nonyl-β-D-glucopyranoside; OM, n-octyl-β-D-maltopyranoside; DM, n-decyl-β-D-maltopyranoside; UDM, n-undecyl-β-D-maltopyranoside; DDM, n-dodecyl-β-D-maltopyranoside; TDM, n-tridecyl-β-D-maltopyranoside; C8E4, polyoxyethylene (4) octyl ether; C12E8, polyoxyethylene (8) dodecyl ether; C12E9, polyoxyethylene (9) dodecyl ether; C10DAO, n-decyl-N,N-dimethylamine-N-oxide; LDAO, n-dodecyl-N,N-dimethylamine-N-oxide; LAPAO, 3-laurylamido-N,N-dimethylpropylaminoxide; FC14, n-tetradecylphosphocholine; MEGA10, decanoyl-N-methylglucamide; DHPC, 1,2-diheptanoyl-sn-glycero-3-phosphocholine.
Mentions: As mentioned above, a detergent that is well-suited for protein solubilization may not be the detergent of choice for crystallization, and detergents can be exchanged through dialysis or during purification while the target protein is bound to a chromatographic column. Just as with the screening of detergents for the optimization of solubilization, a variety of detergents should be screened during the course of crystallization trials. An impressive range of detergents has been used to obtain crystals yielding high-resolution diffraction (Figure 2). From crystal-packing considerations, detergents with the potential to yield the smallest possible micelle region on the solubilized protein should best support the formation of the protein-to-protein contacts needed for crystallization. A potential downside is that smaller detergents tend to have higher CMCs, requiring higher concentrations to maintain protein solubility, and are more likely to destabilize native structure. The goal should therefore be to identify the smallest detergents that maintain homogeneous and monodisperse solutions of structurally sound target proteins. Alternatively, the use of secondary detergents or amphiphiles as additives to alter the properties of mixed micelles has also yielded high-quality crystals [27,28]. The location, or mere presence, of an affinity tag may play a role in determining whether a protein will crystallize, and it may be advisable to conduct crystallization trials on target proteins in which the polyhistidine affinity tags have been removed. Cleavage sites engineered into the expressed protein can be used to remove these tags using a detergent-resistant protease.

Bottom Line: Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts.There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

View Article: PubMed Central - HTML - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

ABSTRACT
Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts. There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.

Show MeSH