Much of the regulation of biological processes is built on the controlled use of protein/protein interactions. By altering these in response to various cues, changes can be produced in protein conformations (via in cis interactions) or the composition of multiprotein complexes (in trans interactions). Either type of these changes can be harnessed to affect signaling through various intracellular pathways.
To investigate natural regulation in these systems, and to potentially lay the foundation for artificial control, we are studying two general types of proteins:
Sensors. We use a combination of biophysics and biochemistry to study two types of photosensory protein domains, the LOV (Light-Oxygen-Voltage) and BLUF (Blue Light sensors Utilizing Flavin) families. Members of both families bind flavin chromophores within small mixed alpha/beta folds, sensitizing them to blue light. However, differences in photochemistry and the types of proteins that they control suggest that there may be important mechanistic differences between the two. With NMR spectroscopy and other biophysical measurements, we have been able to show that illumination of both types of domains lead to changes that are transmitted to within the sensory domain and beyond (Harper et al., 2003; Yao et al., 2008; Wu et al., 2008). Mutations that artificially and constitutively trigger these changes have effects in full length proteins (Harper et al., 2004), validating our in vitro studies on isolated domains. We are continuing this work by examining the roles of critical residues in the transmitting these light-initiated signals and sending them to a wide range of effectors, and further in seeing how generally these are conserved with sensors of other stimuli using the same types of domains (Lee et al., 2008).
Transcription factors. The photosensory LOV domains mentioned above are members of a much larger group of protein/protein interaction domains, the PAS (Per-ARNT-Sim) family. Among other places, these are found within several eukaryotic transcription factors, including members of the bHLH-PAS family (e.g. hypoxia inducible factor (HIF) (Erbel et al., 2003), NPAS2; Holdeman and Gardner, 2001) and the Neurospora white collar circadian clock system (He et al., 2003). In these proteins, PAS domains are critical for building heterodimeric complexes that can bind DNA and activate transcription. We are studying PAS domains from several of these factors, particularly HIF, to investigate the structural basis of specificity in their protein/protein interactions. We have provided the first structural data on dimerization from this system (Erbel et al., 2003; Card et al. 2005) and have worked with Rick Bruick?s group to demonstrate that these data are relevant to full length HIF in living cells (Yang et al., 2005). Notably, these proteins use protein/protein interactions analogous to those used in the LOV-based photosensors (Harper et al., 2003; Erbel et al., 2003), without involving any internally-bound cofactors. We are extending this research to further define these interactions and to investigate parallels with the photosensors that may suggest routes to artificial regulation.
RESEARCH INTERESTS
structural biology
signaling
allostery
artificial regulation
cancer
RECENT PUBLICATIONS
Card, P.B., Erbel, P.J.A. and Gardner, K.H., "Structural basis of ARNT PAS-B dimerization: Use of a common β-sheet interface for hetero- and homodimerization" J. Mol. Biol., 353:664-677, 2005
Yang, J., Zhang, L., Erbel, P.J.A., Gardner, K.H., Ding, K.M., Garcia, J.A. and Bruick, R.K., "Functions of the Per/ARNT/Sim (PAS) domains of the hypoxia inducible factor (HIF)" J. Biol. Chem., 280:36047-36054, 2005
Lee, J., Tomchick, D.R., Brautigam, C.A., Machius, M., Kort, R., Hellingwerf, K.J. and Gardner, K.H., "Changes at the KinA PAS-A dimerization interface influence histidine kinase function" Biochemistry, 47:4051-4064, 2008
Yao, X., Rosen, M.K. and Gardner, K.H., "Estimation of the available free energy in a LOV2-Jα photoswitch" Nature Chemical Biology, 4:491-497, 2008
Q. Wu, W.-H. Ko, K.H. Gardner, "Structural requirements for key residues and auxiliary portions of a BLUF domain" Biochemistry, in press
SIGNIFICANT PUBLICATIONS
Harper, S.M., Neil, L.C. and Gardner, K.H., "Structural basis of a phototropin light switch" Science, 301:1541-1544, September 2003
Erbel, P.J.A., Card, P.B., Karakuzu, O., Bruick, R.K. and Gardner, K.H., "Structural basis of PAS domain heterodimerization in the basic helix-loop-helix-PAS transcription factor hypoxia-inducible factor" Proc. Natl. Acad. Sci., 100:15504-15509, December 2003
Harper, S.M., Neil, L.C., Day, I.J., Hore, P.J. and Gardner, K.H., "Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy" J. Am. Chem. Soc., 126:3390-3391, 2004
Card, P.B., Erbel, P.J.A. and Gardner, K.H., "Structural basis of ARNT PAS-B dimerization: Use of a common β-sheet interface for hetero- and homodimerization" J. Mol. Biol., 353:664-677, 2005
Yao, X., Rosen, M.K. and Gardner K.H., "Estimation of the available free energy in a LOV2-Jα photoswitch" Nature Chemical Biology, 4:491-497, 2008
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