Project Details

Description

Energy coupling is a fundamental biological process that involves the conversion of chemical energy to mechanical work. It is critical for active transport systems; yet, the mechanics underlying this process are poorly understood. The long term objective of this project is to determine a structural basis for coupling ATP hydrolysis to H+ transport by the P- type plasma membrane H+-ATPase of yeast. The stalk region is proposed to mediate coupling by linking the cytoplasmic ATP binding/hydrolysis domain to transmembrane segments involved in proton binding and release. Prominent structural changes in the ATP binding/hydrolysis domain occur during catalysis and an understanding of how these dynamic changes are transmitted through the stalk region and then to the transmembrane segments is a primary goal. The specific aims of this project are designed to define the structural organization of the stalk and nucleotide binding domain by testing specific models, and then identify local changes occurring during the catalytic cycle. Genetic and biochemical probing of these regions will be used to examine critical residues and local structures through directed mutagenesis, suppressor analysis, chemical labeling, cross-linking, and metal catalyzed cleavage studies. Special emphasis will be placed on stalk elements 4 and 5 which flank key regions of the nucleotide binding domain and are directly linked to transmembrane segments engaged in ion binding. Partially uncoupled mutants and low energy substrates will be used to distinguish between structural changes that promote coupled proton transport and those that do not. To better understand how structural changes impact proton translocation, current-voltage analysis will be used to evaluate partial reactions of the transport pathway. In the end, a working scheme for coupling ATP hydrolysis to proton transport will be developed that describes the critical structural changes occurring in the nucleotide binding domain and stalk region following nucleotide binding.
StatusFinished
Effective start/end date1/1/9011/30/04

Funding

  • National Institute of General Medical Sciences: $409,611.00
  • National Institute of General Medical Sciences: $386,356.00
  • National Institute of General Medical Sciences: $421,762.00
  • National Institute of General Medical Sciences: $397,813.00

ASJC

  • Genetics
  • Molecular Biology
  • Structural Biology
  • Biochemistry
  • Catalysis

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