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| [[Image:1a40.gif|left|200px]] | | {{Seed}} |
| | [[Image:1a40.png|left|200px]] |
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| {{STRUCTURE_1a40| PDB=1a40 | SCENE= }} | | {{STRUCTURE_1a40| PDB=1a40 | SCENE= }} |
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| '''PHOSPHATE-BINDING PROTEIN WITH ALA 197 REPLACED WITH TRP'''
| | ===PHOSPHATE-BINDING PROTEIN WITH ALA 197 REPLACED WITH TRP=== |
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| ==Overview==
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| Stringent specificity and complementarity between the receptor, a periplasmic phosphate-binding protein (PBP) with a two-domain structure, and the completely buried and dehydrated phosphate are achieved by hydrogen bonding or dipolar interactions. We recently found that the surface charge potential of the cleft between the two domains that contains the anion binding site is intensely electronegative. This novel finding prompted the study reported here of the effect of ionic strength on the equilibrium and rapid kinetics of phosphate binding. To facilitate this study, Ala197, located on the edge of the cleft, was replaced by a Trp residue (A197W PBP) to generate a fluorescence reporter group. The A197W PBP-phosphate complex retains wild-type Kd and X-ray structure beyond the replacement residue. The Kd (0.18 microM) at no salt is increased by 20-fold at greater than 0.30 M NaCl. Stopped-flow fluorescence kinetic studies indicate a two-step binding process: (1) The phosphate (L) binds, at near diffusion-controlled rate, to the open cleft form (Po) of PBP to produce an intermediate, PoL. This rate decreases with increasing ionic strength. (2) The intermediate isomerizes to the closed-conformation form, PcL. The results indicate that the high specificity, affinity, and rate of phosphate binding are not influenced by the noncomplementary electronegative surface potential of the cleft. That binding depends almost entirely on local dipolar interactions with the receptor has important ramification in electrostatic interactions in protein structures and in ligand recognition.
| | The line below this paragraph, {{ABSTRACT_PUBMED_9865949}}, adds the Publication Abstract to the page |
| | (as it appears on PubMed at http://www.pubmed.gov), where 9865949 is the PubMed ID number. |
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| | {{ABSTRACT_PUBMED_9865949}} |
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| ==About this Structure== | | ==About this Structure== |
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| [[Category: Mutagenesis]] | | [[Category: Mutagenesis]] |
| [[Category: Phosphate-binding protein]] | | [[Category: Phosphate-binding protein]] |
| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 09:46:43 2008'' | | |
| | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 15:58:58 2008'' |
