Bacterial protein toxins are typically produced as water-soluble proteins. However, many of these toxins exert at least some of their effects at the target membrane. These proteins must therefore possess characteristics of both water-soluble and membrane proteins. It then follows that these proteins are often required to undergo large conformational changes in order to exert their toxic mode of action. Understanding the energetics and molecular details of these conformational changes is critical to elucidating the mode of action of protein toxins, with the goals of improving toxin activities (as in the cases of Bt toxins applied to crop biotechnology or immunotoxin improvement for disease therapies), or inhibiting toxin activities (as in the cases of disease management or bioterrorism prevention). In the following sections, we will briefly introduce methods for studying protein conformational changes, followed by a section on factors contributing to the stabilization of protein structures. These introductory sections will then be followed by discussions on soluble protein folding, membrane protein folding, and finally an integration of these two models into a model for understanding bacterial protein toxin conformational changes.
In a perfect scenario for studying the conformational changes of a protein, the three-dimensional structure of each of the relevant states is known. However, in the real world, this is rarely the case. For many proteins, the detailed structure of the native
Central Questions in Protein Toxin Research, Organized by Toxin Life Stage with Toxin Examples3
Central Question or Theme w
Bordetella pertussis adenylate cyclase toxin1
Terminal toxic event
Ca-dependent quaternary structure
Secondary Localization Modification
Ca-dependent membrane insertion
Repeats in Toxin Family (RTX)
ACh receptor binding
Tumor necrosis factor binding
N-terminal Tertiary domain stability assignment
Tol B interaction site
Translocation signal domain
DNA hydrolysis i ot e n
H. pylori vacuolating toxin VacA7-11
Solution structure, localization7 Terminal toxic action11
Structural alteration, localization
Variable oligomeric morphology7 Secondary structure-function assignment11 Quaternary structure determinants11
Acid activation domains11
Lipid-induced conformational change
Yeast K1 viral Cell recognition9 toxin9-13
Clostridium difficile toxin B10
E. coli cytotoxic necrotizing factor l12
Vibrio cholerae toxin14
Solution structure, terminal toxic event12
Numbers refer to chapter references.
A/B toxin, ribosome inactivating toxin
Ion channel activation and pore formation
Disrupted H+ transport
Rho-GTPase Inactivation binding of GTPase protein toxin causing actin depolymeri-
Rho-GTPase toxin zation
protein is known from either x-ray crystallographic data or solution proton nuclear magnetic resonance (NMR) experiments. In some cases, structural information from both techniques is known. NMR-derived structural information is richer than x-ray crystal structure data in that the former also yields insights into the conformational dynamics around the equilibrium structure. NMR can also be used to gather information about protein intermediates, either under native state conditions or by manipulating solution conditions to favor a particular state. Although there is a plethora of structural information for proteins in their native states, to date the detailed molecular structure of a denatured protein has yet to be described. This is undoubtedly due to the conformational heterogeneity of the unfolded state, which hampers structure determination via protein crystallography or NMR spectroscopy. Some general features of unfolded proteins have been described using other biophysical tools such as circular dichroism (CD) spectroscopy or hydrogen exchange measured by NMR.
The absence of detailed structural information does not preclude one from gaining important insights into the conformational changes related to protein function. The only requirement for monitoring protein conformation is a measurable property of the protein that is related to one particular protein state. Proteins have a few intrinsic properties that are suitable for just such observation. Protein secondary structure is a useful indicator of protein conformation and can be monitored using CD spec-troscopy. The loss of protein secondary structure is an indication of the transition from a structured protein state to a less-structured unfolded state. These types of structural alterations may play an important role in the mode of action for a given protein toxin. CD spectroscopy reports on the overall protein conformation. Other spectroscopic techniques such as fluorescence or electron paramagnetic resonance (EPR) spectroscopy can be used to gather more site-specific information. However, to get the more detailed site-specific information, specific residues in the protein are labeled with probes, fluorophores, or paramagnetic molecules. Signals from these incorporated probes can then be followed in in vitro assays that follow specific steps in the mode of action such as protein binding, pore formation, etc. Using a variety of biophysical tools allows one to collect a diverse set of complementary data that can be used to identify localized regions of the toxin that play critical roles in the mode of action of the protein.
Most soluble proteins are marginally stable, typically 3-10 kcal/mol. Therefore, the forces that contribute to stability are balanced delicately near the transition from folded to unfolded protein. The types of interactions that are responsible for protein stability and conformation can be generally classified as hydrophobic or polar, with hydrogen bonding or electrostatic interactions comprising the polar interactions. There is no consensus regarding which of these forces plays the predominant role in protein stability; this has been debated for almost 70 years. Early on, it was suggested that the hydrophobic effect was the primary determinant of protein stability and that hydrogen bonding was at best neutral, or likely destabilizing. In more recent times, hydrogen bonding has come to be viewed as a potentially stabilizing force in maintaining protein structure. Given the marginal stability of proteins in general, it is probably true that for any given protein hydrophobicity may predominate, whereas in a different protein hydrogen bonding may predominate.
Studies on protein stability have led to the general observation that hydrogen bonding can provide more specificity to protein structure than hydrophobic interactions. Mutational studies have shown that proteins are able to slightly adjust local conformation to compensate for changes in hydrophobic packing interactions with marginal effects on protein stability and overall structure. Hydrogen bonds provide specificity due to the directional nature and geometric constraints of a hydrogen bond. Recent studies have shown that a specific side-chain hydrogen bond can provide structural specificity to transmembrane helix interactions.14 In terms of protein toxin mode of action, both hydrophobic and hydrogen bonding interactions can play significant roles in determining the structural interactions that produce the toxic effect on the target site(s) in the cell.
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