..:: Cavanagh lab ::..

Cavanagh Lab Research Topics (click on the title for a more detailed description)

* Bacterial Competence

Approximately 10% of bacteria are naturally competent. Competence refers to the ability of a cell to bind and internalize exogenous DNA, providing an efficient mechanism for the exchange of genetic information. We are studying the critical trigger mechanism for establishing competence in B. subtilis. In B. subtilis, the protein ComK is absolutely necessary for the transcription of late competence genes, and the regulation of ComK stability is regarded as the crucial event in the development of competence. The competence genes encode a set of proteins responsible for the uptake and internalization of transforming DNA. In its non-competent state B. subtilis contains a large protein complex which effectively ties up ComK so it is unable to perform its regulatory functions. The protein complex consists of the proteins ClpC, ClpP, MecA and, of course, ComK. In response to increased cell density, a small protein, ComS, is synthesized. ComS then targets MecA, interacting with its N-terminal domain and breaks up the protein complex. The subsequent release of ComK allows the cell to then become competent. Working with the group of Dr. Dave Dubnau at the Public Health Research Institute (NYC) we are studying the structure/dynamics-function relationships of the MecA protein, the role of ComS and the complex of MecA:ComS. We are particularly interested in any conformational changes that cause the releases of ComK. Particularly interesting is that ComS, in its unbound state, has no discernible structure.

* Transition State Regulators - DNA binding proteins

When bacteria encounter any sort of stress they initially assess whether they should protect themselves. If the stress is persistent, a large morphological change is undertaken as the cell strives to survive. At first the bacteria enter a so-called transition state where processes required for continued growth are regulated simultaneously with those required for protection. Many processes are controlled at this time by, surprisingly, only a few regulatory general proteins. These are a relatively new class of proteins known as transition-state regulators. Transition-state regulators are DNA-binding proteins that recognize and interact with an enormous array of gene promoter regions. They are able to target seemingly disparate nucleotide sequences with little effort. We have recently solved the high-resolution structure of the DNA-binding domain of the transition-state regulator, AbrB, from Bacillus subtilis. This domain, noted AbrBN, exhibits a novel DNA-binding motif. Our studies also suggest an as yet uncharacterized DNA-binding mechanism.

* Response Regulators - Control of sporulation

Continued exposure to stress causes the bacteria to go into a more self-protective mode. In our model system, this means the onset of sporulation, induced by a pair of so-called two-component systems. Two-component systems are ubiquitous signal transduction modules involved in a myriad of processes but have found particular notoriety by their critical role in the generation of bacterial pathogenesis and antibiotic resistance. They are found in all bacteria so far studied and are responsible for the ability of the cells to adapt to changing environments and ultimately to protect the bacteria in times of life threatening hostility. At some level they are responsible for the development of all bacterial virulence. They consist of a histidine protein kinase and a response regulator. In times of stress the kinase becomes phosphorylated and subsequently passes the phosphoryl group onto the response regulator, which undergoes a conformational change, thereby allowing it to interact with its next target(s), be it DNA or another protein. Response regulators share similar structures and general function, however their specific mechanism of action is not well understood. Our recent work has defined much about the recognition processes of response regulators, showing that protein structure is only part of the answer. We have shown that millisecond timescale motions are involved in protein:protein interactions for the response regulator Spo0F.

* Calbindin D28K - Calcium binding sensor and buffer protein

Calbindin D28K is a biologically essential calcium-binding protein of unknown tertiary structure that is required for normal neural function. Neural tissues such as Purkinje cells of the cerebellum, peripheral nerve cells, intestinal and dorsal root ganglion cells, photoreceptor and ganglion cells of the eye and cochlear cells express calbindin D28K for normal function. It is co-localized with the plasma membrane calcium pump in neural and ocular tissues. Mice with null or reduced levels of the protein develop ataxia and impaired intracellular calcium homeostasis or have deficits in memory and hippocampal long-term potentiation. Additionally, calbindin D28K is expressed in calcium transporting tissues such as the kidney and intestine, and in islet cells of the pancreas, modulating insulin release in these cells. It is unique among calcium binding proteins in that it consists of 6 EF hands, four of which are involved in metal chelation. The structures of Ca²⁺-free and Ca²⁺-bound calbindin D28K are unknown, as are the conformational effects of binding sequential Ca²⁺ ions and indeed the order in which the EF hands co-ordinate the incoming metal ions. Does the protein undergo sequential smaller conformational changes as the metal ions bind or does it undergo one large conformational change either initially or at some point along the binding pathway? We will address such structural modifications and the determination of the binding by a combination of NMR, mass spectrometry, CD spectroscopy and a variety of other bioanalytical methods.