Research

Microbiology projects

Bacterial photobiology and photoreceptors (refs 6, 7, 10, 21, 22, 26)

Bacterial genome sequencing projects have unexpectedly revealed the presence of photosensory proteins in a many bacteria, including chemotrophs. Most of these photoreceptors are unstudied and have an unknown functional role, indicating that many bacterial photoresponses remain to be uncovered. One of the six classes of photosensory proteins is photoactive yellow protein (PYP).

PYP is a unique photoreceptor that contains a novel p-coumaric acid chromophore. After early work that identified the PYP from H. halophila as the photoreceptor for negative phototaxis, we recently reported that in the deep sea bacterium Idiomarina loihiensis PYP functions to regulate biofilm formation. We also reported six novel members of the PYP family of photoreceptors. Currently we are examining the in vitro properties and in vivo signaling of PYPs from a range of different organisms, and are exploring structure-function relationships in the PYP family.

For more information see http://www.photobiology.info/Hoff.html Figure 1

Figure 1. Negative phototaxis in Halorhodospira halophila. A blue light sport was projected into a motile culture of H. halophila cells places in an optically flat capillary. The cells avoid both the dark and exhibit negative phototaxis to the blue light, thus ending up in a ring around the light spot (26).

Physiology of extreme halophiles

Approximately 97% of all water on earth is present in saline oceans, saline lakes, inland seas, and saline groundwater. Thus, saline and hypersaline environments are highly abundant and of great ecological significance. We are examining halophilic adaptations in Halorhodospira halophila, one of the most halophilic organisms known. This work is guided by our determination of the genome sequence of this organism in collaboration with the DOE JGI (see section 3). Currently we are focusing on the use of different osmoprotectants under various growth conditions.

Figure 2. Electron micrograph of Halorhodospira halophila. Note the two polar flagella. Picture by Dr. Wander Sprenger.

Bacterial genomics and bioinformatics: genome-based studies of physiology (ref 7)

We are using bacterial genomes to better understand bacterial physiology. We are completing an analysis of the genome of H. halophila, an extremophilic and aeaerobic purple photosysnthetic Proteobacterium. The information obtained from the genomic data is guiding our studies on the halophilic adaptations in this organism. We are following a similar approach to the mechanism of photoregulation of biofilm formation in I. loihiensis. In the case of the PYP family of photoreceptors we are mining all available bacterial genomes for information on its signal transduction and in vivo functioning.

Figure 3. Model for signal transduction by PYP in Idiomarina loihiensis (7).

Biochemistry projects

Photoactive yellow protein (refs 1, 2, 4, 5, 10, 11, 12, 15, 22-25)

Photoactive yellow protein (PYP) is a bacterial blue-light receptor and a prototype of the large and diverse PAS domain superfamily of signaling proteins. It exhibits a photocycle based on the photoisomerization of its p-coumaric acid chromophore. We use PYP as a model system to obtain insights into a range of important processes, including protein-chromophore interactions, receptor activation, functional protein dynamics, and protein folding. In addition, we are exploring diversity in the functional properties of PYPs from different organisms.

Figure 4. Structure of PYP with its active site (Borgstahl et al. 1995, Biochemistry 34: 6278-6287)

Receptor activation and signal transduction (refs 3, 6, 7, 14, 17-19, 21)

Our studies of the light-induced structural changes in PYP revealed that receptor activation of this receptor involves partial protein unfolding. We are currently exploring the role of this partial unfolding process in signal relay by PYP. We found that proton transfer at the active site of PYP is a critical step in its activation. This has resulted in a novel model for receptor activation, in which light-induced intramolecular proton transfer causes the formation of a destabilizing buried charge, the “electrostatic epicenter”, which triggers large conformational changes, the “protein quake”, for signal transduction. We are currently investigating the role of partial unfolding in the signaling of other protein systems.

Figure 5. Model for the photocycle of PYP. The pB intermediate is the proposed signaling state of PYP. Active site structural changes during the photocycle are schematically indicated (2)

Biosensors and biodetection

We are using protein engineering and in vitro evolution to develop applications in which chromophoric proteins act as fluorescent reporters in biosensing applications. Such sensors promise to address open challenges in biodetection for both biosecurity and biomedical applications.

Protein folding (refs 4, 11, 13, 16-18)

The properties of PYP and the link that we discovered in this protein between its light-induced function and partial unfolding render PYP into an attractive model system to study protein folding. We have used stopped-flow rapid mixing to perform a Chevron analysis of PYP, revealing that folding and signaling in PYP share the same pathway. Spectroscopic studies revealed that the signaling state of PYP resembles a molten globule state. More recently, we reported the effect of proline isomerization on the PYP photocyce and used the light sensitivity of PYP to investigate residual structure in the “fully unfolded” state. We are also probing folding and stability in PYP using axis-dependent single molecule force spectroscopy (see section 10). Figure 6

Figure 6. Chevron analysis of folding and pB decay in PYP (18).

PAS domains (refs 1, 14, 17)

PYP is a prototype of the large and diverse PAS domain superfamily. The PAS domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. Over 20,000 proteins have been found to contain a PAS domain, with 43 PAS domains present in the human genome. We are using PYP as a model system to understand PAS domain structure and function. Recently, we found that, unexpectedly, the residues that are highly conserved in the PAS domain family are often involved in allowing adequate protein production. We have recently identified Asn43 as a critical residue in PYP that is likely to be important in many PAS domains.

Figure 7. Conservation in the PAS domain superfamily of the hydrogen bonding interactions of the residue corresponding to Asn43 in the PYP from H. halophila.

Biophysics projects

Biophysics and spectroscopy of proteins (refs 1, 2, 5, 8, 13-20, 23, 25)

We are developing and using photoactive yellow protein as a powerful model system to study fundamental processes in the biophysics of proteins. The light-triggered events in PYP make it highly suitable for studies on protein folding, functional protein dynamics, receptor activation and signal transduction, and proton transfer. In addition, we are using a novel axis-dependent approach in single molecule force spectroscopy to experimentally probe the energy landscape for protein folding and function. Based on the high-throughput methods for spectroscopic studies of protein mutants we are examining the robustness and evolvability of proteins.

Both in our laboratory and in collaboration we are using a range of spectroscopic and biophysical techniques to examine PYP and related proteins, particularly UV/vis absorbance, fluorescence, vibrational, ultrafast, and force spectroscopy. We are particularly interested in developing the use of Fourier transform infrared spectroscopy for time-resolved structural biology in collaboration with Prof. Aihua Xie. We are are preparing a set of specifically isotopically labeled proteins to aid in this development.

Figure 8. Time-resolved FTIR spectroscopy of pB decay in the PYP from I. loihiensis (7).

Single molecule force spectroscopy (refs 3, 9, 13, 14)

The development of techniques for single molecule studies is providing an exciting novel tool for studies of biomolecules. We have developed an approach in which two Cys residues are introduced at surface exposed positions in a small single-domain protein, resulting in the formation of a poly-protein linked by disulfide bonds. Such polyproteins are excellent substrates for single molecule force spectroscopy. By placing the Cys residues at different locations it is possible to perform axis-dependent force spectroscopy. Our atomic force microscopy studies on two different axes in photoactive yellow protein in the dark and in the light have yielded two insights. First, a 3 nm reduction in unfolding length was observed along both axes, allowing the localization of partial unfolding to the PAS core of PYP. Secondly, we unexpectedly found that while folding along one axis proceeds cooperatively, the other axis exhibits non-cooperative unfolding. Thus, the same protein can exhibit strong anisotropy in its unfolding mechanism.

Figure 9. Axis-dependent single molecule force spectroscopy of PYP using atomic force microscopy (13, 14).

Protein-chromophore interactions: spectral tuning and pKa tuning (refs 1, 2, 5, 10, 12, 23)

Protein-ligand interactions that result in the tuning of active site groups to functionally relevant values is an important theme in biochemistry. We are using PYP to probe spectral tuning and pKa tuning. The mechanism responsible for spectral tuning is a classic problem in photobiology. Spectral tuning is illustrated by our color vision, in which the same retinal can absorb in the blue, green, or red depending on the amino acid sequence of the rhodopsin that the retinal chromophore is bound to. The issue of pKa tuning is of general importance for understanding catalysis and proton transfer in proteins. We have reported a number of PYP mutants in which the pKa and absorbance maximum is significantly shifted. Based on a combined analysis of the absorbance and fluorescence emission spectra of these mutants, we have found a novel mechanism for spectral tuning in which the width of the energy surface of the electronically excited state is altered. This provides a novel approach to understanding spectral tuning.

Figure 10. Absorbance and fluorescence emission spectra of the E46X library of mutants (5).

High-throughput protein biophysics and protein structure-function relationships (refs 1, 5, 12)

An important current trend in the life sciences is the development of application of high-throughput methods for applications ranging from DNA sequencing to screening drugs and protein structure determination. We have developed high-throughput methods for the purification and spectroscopic characterization of PYP mutants. This work revealed that PYP combines a high level of robustness against point mutations with a high level of evolvability: a typical mutation will not abolish the function (yellow color and photoactivity) of PYP, but it will alter its functional properties.

Figure 11. Low pH titration curves of the E46X library of PYP mutants in 96-well format (12). The bsorbance spectra of wtPYP and its E46Q and E46I mutants are also shown.

Experimental techniques and equipment in the Hoff lab

Molecular genetics and site-directed mutagenesis

DNA gel boxes and Alpha-Innotech gel documentation system
A PerkinElmer Geneamp2400 thermocycler
Protein overexpression and purification
New Brunswick incubator/shakers
Sorvall Evolution RC centrifuge
Beckmann-Coulter Allegra X-12R tabletop centrifuge
Akta FPLC system with autoinjection
UV/vis and fluorescence spectroscopy
HP8453 diode-array absorbance spectrophotometer
Cary 300 absorbance spectrophotometer.
Fluoromax-3 fluorimeter (Spex – Horiba Jobin Yvon) with Peltier element.
Rapp OptoElectronic DM-10X flash lamp system (> 35 mJ per flash)
Cuda lamp with Uniblitz optical shutter
Stopped-flow rapid mixing
SX-18MV stopped-flow spectrometer, 1 ms time resolution (Applied Photophysics).
RX.2000 stopped-flow accessory (Applied Photophysics).

Microscopy

Nikon Eclipse 80i dark field and fluorescence microscope with motion analysis. FTIR spectroscopy Equipment: collaboration with Aihua Xie
Single-molecule force spectroscopy Equipment: OSU core facility AFM and collaboration with Norber Scherer
Bacterial physiology [picture of H. halophila growing] Chemical composition of cells in core facility