STRUCTURE 1BM0
From Proteopedia
| Line 11: | Line 11: | ||
| - | ==Crystallization of the tetragonal crystal== | + | ==Crystallization and structural determination of the tetragonal crystal== |
| - | pHSA purchased from sigma chemical co. was crystallized using the hanging drop method. Yellow colored crystals were obtained from a solution containing 150-255 mg/ml protein, 50mM potassium phosphate, 30-38% PEG 400 and 5mM sodium azide. The crystals belonged to tetragonal space group P4212 with cell dimensions a=187.1A°, c=80.5A°. Crystals | + | pHSA purchased from sigma chemical co. was crystallized using the hanging drop method. Yellow colored crystals were obtained from a solution containing 150-255 mg/ml protein, 50mM potassium phosphate, 30-38% PEG 400 and 5mM sodium azide. The crystals belonged to tetragonal space group P4212 with cell dimensions a=187.1A°, c=80.5A°. Crystals reach a resolution of 3.0A° due to high solvent concentration. |
| - | + | ||
| - | + | ||
Intensity data was collected using Rigaku R-AXIS 11c area detector on a rigaku RU-200H x-ray generator. The diffraction data was processed with PROCESS. Hgcl2 was used for phasing with multiple isomorphous replacement. The heavy atom sites were located using the HASSP program. The phasing statistics can be seen in table 1. | Intensity data was collected using Rigaku R-AXIS 11c area detector on a rigaku RU-200H x-ray generator. The diffraction data was processed with PROCESS. Hgcl2 was used for phasing with multiple isomorphous replacement. The heavy atom sites were located using the HASSP program. The phasing statistics can be seen in table 1. | ||
| - | ==crystallization of the triclinic crystal== | + | ==crystallization and structural determination of the triclinic crystal== |
Before crystallization fatty acids were removed from the sample solution using activated powdered charcoal. The fractions were obtained by elution using NACL salt gradient. All the main peak fractions were pooled together and concentrated by ultra filtration followed by dialysis. The purified protein was crystallized using hanging drop method. | Before crystallization fatty acids were removed from the sample solution using activated powdered charcoal. The fractions were obtained by elution using NACL salt gradient. All the main peak fractions were pooled together and concentrated by ultra filtration followed by dialysis. The purified protein was crystallized using hanging drop method. | ||
| - | |||
| - | ==structural determination of the triclinic crystal== | ||
Rigaku R-AXIS IIc was used to measure the diffraction data of the triclinic crystal. Molecular replacement and crystallographic refinement of the pHSA triclinic crystal was implemented in X-PLOR. The crystallographic R factor of the current atomic model is 21.8 and 28.2% respectively. The atomic co-ordinates and structure factors of triclinic pHSA and rHSA have been deposited in the Brookhaven protein data bank. | Rigaku R-AXIS IIc was used to measure the diffraction data of the triclinic crystal. Molecular replacement and crystallographic refinement of the pHSA triclinic crystal was implemented in X-PLOR. The crystallographic R factor of the current atomic model is 21.8 and 28.2% respectively. The atomic co-ordinates and structure factors of triclinic pHSA and rHSA have been deposited in the Brookhaven protein data bank. | ||
Revision as of 00:36, 7 March 2010
Contents |
Abstract
The three dimensional structures of pHSA and rHSA triclinic crystals were determined at 2.5A°resolution through multiple isomorphous replacement with a known tetragonal crystal. The pHSA and rHSA structures are similar with an r.m.s deviation of 0.24 A° for all the cα atoms. Cys 34 does not have a disulfide link with any ligand. A pocket of hydrophobic and positively charged residues is formed at domains 2 and 3 where a wide range of compounds can be accommodated. The surface of the domain has three binding sites for long chain fatty acids.
Introduction
HSA has a blood concentration of 5g/100ml and is the most abundant protein in the blood plasma. HSA has a high affinity for cu+2, zn+2, fatty acids , amino acids , metabolites and many other drug compounds. The protein brings solutes in the blood to the target organs and also maintains the PH and osmotic pressure of the plasma hence it is used as a carrier of drugs to their targets. HSA has 585 residues with 17 disulfide bridges and one free cysteine. Many reports contained crystal forms of HSA but none provided structural information. This article discusses the crystal form of defatted HSA and the molecular aspects of the protein are determined at the highest resolution.
Crystallization and structural determination of the tetragonal crystal
pHSA purchased from sigma chemical co. was crystallized using the hanging drop method. Yellow colored crystals were obtained from a solution containing 150-255 mg/ml protein, 50mM potassium phosphate, 30-38% PEG 400 and 5mM sodium azide. The crystals belonged to tetragonal space group P4212 with cell dimensions a=187.1A°, c=80.5A°. Crystals reach a resolution of 3.0A° due to high solvent concentration. Intensity data was collected using Rigaku R-AXIS 11c area detector on a rigaku RU-200H x-ray generator. The diffraction data was processed with PROCESS. Hgcl2 was used for phasing with multiple isomorphous replacement. The heavy atom sites were located using the HASSP program. The phasing statistics can be seen in table 1.
crystallization and structural determination of the triclinic crystal
Before crystallization fatty acids were removed from the sample solution using activated powdered charcoal. The fractions were obtained by elution using NACL salt gradient. All the main peak fractions were pooled together and concentrated by ultra filtration followed by dialysis. The purified protein was crystallized using hanging drop method. Rigaku R-AXIS IIc was used to measure the diffraction data of the triclinic crystal. Molecular replacement and crystallographic refinement of the pHSA triclinic crystal was implemented in X-PLOR. The crystallographic R factor of the current atomic model is 21.8 and 28.2% respectively. The atomic co-ordinates and structure factors of triclinic pHSA and rHSA have been deposited in the Brookhaven protein data bank.
Results
Structural determination and quality
The structure of HSA was determined using to two different crystal forms with complementary properties. For solving the structure of protein, isomorphous replacement techniques were employed. pHSA consists of two HSA molecules each containing 5-582 residues and 7 water molecules whereas rHSA contains 5-282 residues and four water molecules. Due to conformational flexibility the electron density of residues 1-4 and 583-585 is not clearly observed. The Ramachandran plot reveals that all the residues fall into either the energetically favorable and allowed regions. The average temperature factors differ from residue to residue.
Local symmetry in the triclinic lattice
The asymmetric unit of the triclinic crystal consists of two HAS molecules. The local twofold symmetry is strict with a rotational angle of 179.8 degrees and translation vector length of less than 0.15 A for both pHSA and rHSA. The two molecules have identical structures with r.m.s deviations at c alpha, backbone atoms and the hydrogen atoms of 0.28,0.31 and 0.66.
Overall structure
The structures of pHSA and rHSA are identical with an r.m.s deviation of 0.24A° at the c alpha atoms.HSA is a heart shaped protein with dimenasions of 80 x80x 30 A° consisting of three domains I,II, and III. Each domain is further divided into two sub-domains a and b.The shapes of domain II and III are similar. Domains I and II are perpendicular to each other hence forming a T shape and domain III forms a Y shape with the domains I and II. Due to this arrangement the protein has a heart shape. The HSA molecule shows molecular flexibility in different conditions due to relative motion of its domains.
Subdomains
Free sulfhydryl group at cys34
Binding sites for drugs and other compounds
Conclusion
References
Proteopedia Page Contributors and Editors (what is this?)
Bhagiradhi Somalanka, Irene Becerra, Neeharika Pothuri, Michal Harel, Valerie Orovwigho
