BIOCHEMICAL AND CRYSTALLOGRAPHIC STUDIES OF RECEPTOR AND MEMBRANE PROTEINS 
Sherry L. Mowbray, Ph.D., Assistant Investigator 
Dr. Mowbray is using the tools of biochemistry 
and x-ray crystallography to study the function of 
receptors and membrane proteins. 
I. Receptors in Bacterial Chemotaxis. 
Chemotaxis is the process by which motile cells 
are able to respond to concentration gradients of 
chemicals in their environment. The genetics and 
behavior of the system have been well studied in 
the gram-negative b^LCtena. Escherichia coli and Sal- 
monella typhimurium. In these cells, two types of 
receptors, soluble ones from the periplasm and 
membrane-bound ones from the cytoplasmic mem- 
brane, are involved in interpreting and conveying 
environmental data to the motor machinery of the 
cell. Some attractants bind to a specific periplasmic 
protein, which then interacts with the appropriate 
membrane receptor. Other attractants are capable 
of binding directly to the membrane receptor pro- 
teins. In both cases it is the membrane receptor 
that carries the information across the cytoplasmic 
membrane and converts it into usable data for the 
cell. The membrane receptors are also involved in 
adaptation to binding signals that persist, through a 
system that involves covalent modification (methyl- 
ation) of the internal portions of the membrane re- 
ceptors. 
II. Membrane Receptors. 
The relationship between the response systems 
that utilize periplasmic proteins and those that do 
not is being studied to learn about the mechanisms 
of transmembrane signaling. The main subject of 
this work has been the aspartate receptor, a mem- 
brane protein that directs response of E. coli to 
both maltose (when it is bound to a periplasmic 
binding protein) and aspartate (by direct binding). 
The responses to maltose and aspartate have been 
shown to be additive and independent. The re- 
sponse to one will occur whether or not the bacte- 
ria have been previously adapted to the other. The 
addition of both attractants simultaneously gives 
rise to a signal that is larger than that to either of 
the stimulants alone. These responses have been 
shown to channel eventually into the same path- 
ways for signaling and adaptation. 
Three specific arginine residues in the external 
portion of the receptor have been shown to be in- 
volved in binding of aspartate. Mutations at these 
residues affect the binding of aspartate to differing 
degrees, depending on the site and, to some de- 
gree, on the residue introduced. In addition, re- 
placement of at least one of the arginines with a ly- 
sine did not impair transmission of the aspartate 
signal. Another of the sites could not accept lysine 
as a replacement. Therefore, a positively charged 
environment (probably a pocket) appears to be an 
essential, but not sufficient, determinant of binding 
of the negatively charged aspartate to its receptor. 
These mutations also had effects on the maltose re- 
sponse, which varied with the site altered. These 
observations and the earlier data on independence 
have led to an alternate proposal that maltose 
(binding protein) sends an attractant signal, be- 
cause its binding site is located on the signaling 
pathway for aspartate, and that the three positive 
charges are critical to proper signal transmission, 
rather than directly forming an aspartate-binding 
site. These models are being further tested. 
The aspartate receptor from 5. typhimurium has 
been purified to homogeneity in detergent solu- 
tion, and the laboratory is currently attempting 
crystallization of this protein. 
III. Periplasmic Receptors of Bacterial Chemotaxis 
and Transport. 
The periplasmic proteins are also the primary re- 
ceptors for transport of the bound compounds 
through distinct membrane systems. Three of these 
proteins, the glucose/galactose, ribose, and dipep- 
tide receptors, are being used to study the activa- 
tion and recognition processes involved in chemo- 
taxis and transport. 
The ribose and glucose/galactose receptors com- 
pete for binding of a membrane chemotaxis recep- 
tor. The structure of the glucose/galactose protein 
from S. typhimurium has been solved in this labo- 
ratory to 2.4 A resolution by the method of multi- 
ple isomorphous replacement. The correlation of 
the structure with the sequence similarities be- 
tween these two receptor sequences has suggested 
regions of the protein that are probably involved in 
chemotactic function. 
An improved method of large-scale purification of 
the binding proteins has been developed and has 
allowed purification of ribose receptor. The ribose 
receptors from 5. typhimurium and E. coli have 
Continued 
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