Characterization of the Mechanosensitive Channel of Large Conductance

Citation

Strop, Pavel (2002) Characterization of the Mechanosensitive Channel of Large Conductance. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/nkqb-gd95. https://resolver.caltech.edu/CaltechETD:etd-05092002-155216

Abstract

Osmoregulation is an essential process in bacteria and higher organisms regulated by the mechanosensitive ion channels. The mechanosensitive channel of large conductance (MscL) is an integral membrane protein that responds to pressure in an effort to prevent cell lysis during osmotic shock. Conversion of MscL from a membrane bound form to a water soluble form was attempted by three methods: computational design, random mutagenesis and chemical modification. The water soluble form of MscL was achieved with cysteine modification method. The stability, pH dependence, and C-terminal helix of MscL were also investigated.

The structure of the cab beta-class carbonic anhydrase (Cab) has been determined to 2.1 A resolution. Cab exists as a dimer with a fold similar to plant beta-class carbonic anhydrases. The active site zinc is coordinated by Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The difference between plant and cab beta-class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes molecule near the active site, suggesting a proton transfer pathway to the solvent.

The structure of the nitrogenase iron protein in the all-ferrous [4Fe-4S]0 form has been determined to 2.2 A resolution. The structure demonstrates that major conformational changes are not necessary to accommodate cluster reduction to the [4Fe-4S]0 state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the capability of existing in three oxidation states.

The role of surface salt bridges in protein stabilization has been investigated. The NMR structure of a rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges is presented here. The analysis shows that the surface sidechain to sidechain salt bridge between does not stabilize PFRD-XC4. The mainchain to sidechain salt bridge, however, stabilizes PFRD-XC4 by 1.5 kcal mol-1. The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.

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