Dr. Hoff

Research in the laboratory of Wouter D. Hoff

Summary of research interests and ongoing projects

We use photoreceptor proteins as model systems for studying protein folding, function, and signaling, and have started to explore biotechnological applications for these proteins. Our long-term goal is to uncover fundamental principles in these processes, and to develop biosensors with useful applications. This research uses a wide range of approaches, from genomic studies to molecular genetics, protein biochemistry, protein spectroscopy and biophysics. We discovered that the activation of photoactive yellow protein (PYP), a bacterial blue-light receptor from the halophilic purple sulfur bacterium Halorhodopsira halophila, involves partial unfolding, providing an unexpected link between signaling and protein folding. We are analyzing the genome from H. halophila, and are performing bioinformatics-guided experiments to understand its extremophilic physiology. We use a range of spectroscopic approaches to unravel the fundamental mechanisms in the activation of PYP, and have developed novel high-throughput protein biophysics approaches to explore protein structure-function relationships. Our single-molecule force spectroscopy measurements allow us to apply a force to unfold the protein along a specific axis, determined by the position of introduced Cys residues. In addition, we are developing novel highly sensitive, specific, and fast biodetection devices based on engineered proteins that allow a direct optical readout.


Current lab members


Dr. Wouter D. Hoff, PI, Associate Professor link to CV Hoff

Dr. Masato Kumauchi, Research Assistant Professor link to CV Kumauchi
Ms. Miwa Hara, PhD Student
Mr. Ratnakar Deole, PhD Student
Ms. Rachana Rathod, PhD Student  

Hoff lab contact information


E-mail: wouter.hoff@okstate.edu

Tel (office): 405-744-4449

Tel (lab): 405-744-4459

Lab location: 230R Henry Bellmon Research Center

Office location: 230A Henry Bellmon Research Center


Mailing address:

Department of Microbiology and Molecular Genetics

Oklahoma State University

307 Life Sciences East

Stillwater, OK 74078-3020


Our work in the news



· On November 2, 2010 the Oklahoman published a news feature on a joint project with Prof. Li Ma at OSU on developing novel biosensors

· Our lab was featured on television (August 13, 2010)

· Philip et al., Proc. Natl. Acad. Sci. USA 107: 5821-5826 was selected for the Faculty of 1000 Biology for 2010

· Our lab was featured on the web site of the OSU College of Arts & Science

· Lee et al., Proc. Natl. Acad. Sci. USA 98, 9062-9067 (2001) was discussed in a commentary article “Shedding light on protein folding” in J. Cell. Biol. 154, 677 (2001).


Research projects and themes in the Hoff lab

Microbiology projects

1. 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).


2. 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

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

3. 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 purople 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

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


Biochemistry projects


4. 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

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


5. 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

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).


6. 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.


7. 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).


8. 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

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


9. 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

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


10. 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

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


11. 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

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


12. 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

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).


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


On-campus collaborations, contacts, and resources:

Prof. Aihua Xie, Department of Physics: Photoactive yellow protein, protein biophysics, and FTIR spectroscopy, biosensors.

Prof. Rob. Burnap , Department of Microbiology and Molecular Genetics: photobiology and bioenergetics.

Prof. Mostafa Elshahed, Department of Microbiology and Molecular Genetics: halophilic adaptations.

Dr. Steve Hartson, Department of Biochemistry and Molecular Biology and Recombinant DNA/Protein Core Facility: DNA sequencing and protein mass spectrometry.

Mr. Terry Colberg, OSU Microscopy Laboratory: electron microscopy.

Key off-campus collaborators:

Dr. Jean Challacombe, Los Alamos National Laboratory in the Genomic sequencing and computational biology group, Los Alamos, NM.

Utilizing bioinformatics tools to better understand the genome of Halorhodospira halophila and the genomic basis of halophilic adaptations in general.


Prof. Klaas J. Hellingwerf, Laboratory for Microbiology, University of Amsterdam, Amsterdam, the Netherlands.

The role of photoactive yellow protein in regulating biofilm formation in Idiomarina. loihiensis.


Prof. Andrzej Joachimiak, Biosciences Division, Argonne National Laboratory, Argonne, IL.

High-throughput X-ray crystallographic structure determination of PYP mutants with altered functional properties.


Prof. Delmar Larsen, Department of Chemistry, University of California – Davis, CA.

Using ultrafast visible and infrared absorbance spectroscopy and two-photon spectroscopy to understand the primary events in the photoactivation of PYP.


Prof. Rich Mathies, the Department of Chemistry, the University of California – Berkeley, CA.

Time-resolved resonance Raman spectroscopy of PYP.


Prof. Douglas Raiford at the Department of Computer Science and Engineering, University of Montana, Missoula, MT.

Genome bioinformatics of halophilic adaptations, with emphasis preferences in on amino acid usage.


Prof. Norbert Scherer at the Department of Chemistry, the University of Chicago, Chicago, IL.

Single molecule studies of protein folding and function.


Dr. Ulrich Krauß and Ms. Kathrin Klein, Institute for Molecular Enzyme Technology, Research Center Jülich, Jülich, Germany.

Technology for fluorescent labeling of enzymes.


Prof. Jianping Wang at Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.

Two-dimensional infrared spectroscopy.


Prof. Yuxiang Weng at the Institute of Physics, Chinese Academy of Sciences, Beijing, China.

Infrared temperature-jump spectroscopy.


Yan Jie, Department of physics, National University of Singapore, Singapore.

Single-molecule magnetic tweezer measurements on proteins.


Prof. Andong Xia at Institute of Chemistry, Chinese Academy of Sciences, Beijing, China.

Fluorescence correlation spectroscopy.


Recent publications:


The work of Dr. Wouter D. Hoff has been quoted over 2,360 times in international scientific literature.

1) Kumauchi M, Kaledhonkar S, Philip AF, Wycoff J, Hara M, Li Y, Xie A, Hoff WD. 2010. A conserved helical capping hydrogen bond in PAS domains controls signaling kinetics in the superfamily prototype photoactive yellow protein. J. Am. Chem. Soc. 132: 15820-15830.

Philip AF, Kumauchi M, Hoff WD. 2010. Robustness and evolvability in the functional anatomy of a PAS domain. Proc. Natl. Acad. Sci. USA 107: 17986-17991.

3) Hoff WD, Kang Z, Kumauchi M, Xie A. 2010. Changes in active site hydrogen bonding upon the formation of the electronically excited state of photoactive yellow protein. In Hydrogen bonding and transfer in the excited state, editors: Zhao G-J and Han K-L, Wiley, in press.

4) Hoff WD. 2010. Single-molecule and nanoscale approaches to biological signaling. In Comprehensive nanoscience and technology Vol. 4, editors: van Grondelle R, Krueger B, Walker G, Elsevier, in press.

5) Lee B-C, Kumauchi M, Hoff WD. 2010 Chromophore isomerization reduces native-like residual structure in the fully unfolded state of the blue light receptor photoactive yellow protein. J. Biol. Chem. 285: 12579-12586.

6) Philip AF, Nome RA, Papadantonakis GA, Scherer NF, Hoff WD. 2010. Spectral tuning in photoactive yellow protein by modulation of the shape of the excited state energy surface. Proc. Natl. Acad. Sci. USA 107: 5821-5826.

7) Hoff WD, van der Horst MA, Nudel CB, Hellingwerf KJ. 2009. Prokaryotic phototaxis. In: Chemotaxis: methods and protocol. Methods in Molecular Biology Vol. 571, pp. 25-49, editors: Jin T and Hereld D, Humana Press.

8) van der Horst MA, Stalcup TP, Kaledhondar S, Kumauchi M, Hara M, Xie A, Hellingwerf KJ, Hoff, WD. 2009. Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis. J. Am. Chem. Soc. 131, 17443-17451.

9) Xie A, Hoff WD. IR spectroscopy illuminates protein structure and function. 2009. BioOptics World Sept/Oct 2009, 18-21.

10) Hoff WD, Spudich JL. 2008. Single-molecule tour de force: teasing apart a signaling complex. Structure 16, 1149-1150.

11) Kumauchi M, Hara M, Stalcup P, Xie A, Hoff WD. 2008. Identification of six new photoactive yellow proteins: diversity and structure-function relationships in a bacterial blue light photoreceptor. Photochem. Photobiol. 84, 956-969.

12) Lee B-C, Hoff WD. 2008. Proline 54 trans-cis isomerization is responsible for the kinetic partitioning at the last-step photocycle of photoactive yellow protein. Protein Sci. 17, 1-10.

13) Philip AF, Eisenman KT, Papadantonakis GA, Hoff WD. 2008. Functional tuning of photoactive yellow protein by active site residue 46. Biochemistry 47, 13800-13910.

14) Nome RA, Zhao JM, Hoff WD, Scherer NF. 2007. Anisotropy in protein unfolding: integrated nonequilibrium single-molecule experiments, analysis, and simulation. Proc. Natl. Acad. Sci. USA 104, 20799-20804.

15) Zhao JM, Lee H, Nome RA, Majid S, Scherer NF, Hoff WD. 2006. Single-molecule detection of structural changes during PAS domain activation. Proc. Natl. Acad. Sci. USA. 103, 11561-11566.


Selected older publications (@ indicates more than 100 citations in Web of Science):


15) Pan D, Philip A, Hoff WD, Mathies R.A. 2004. Time-resolved resonance Raman structural studies of the pB’ intermediate in the photocycle of photoactive yellow protein. Biophys. J. 86, 2374-2382.

16) Lee B-C, Croonquist PA, Hoff WD. 2001. Mimic of photocycle by a protein folding reaction in photoactive yellow protein. J. Biol. Chem. 276, 44481-44487.

17) Lee B-C, Croonquist PA, Sosnick, TR, Hoff WD. 2001. PAS domain receptor photoactive yellow protein is converted to a molten globule state upon activation. J. Biol. Chem. 276, 20821-20823.

18) Lee B-C, Pandit A, Croonquist PA, Hoff WD. 2001. Folding and signaling share the same pathway in a photoreceptor. Proc. Natl. Acad. Sci. USA 98, 9062-9067.

19)@ Xie A, Kelemen L, Hendriks J, White BJ, Hellingwerf KJ, Hoff WD. 2001. Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation. Biochemistry 40, 1510-1517.

20)@ Xie A, van der Meer L, Hoff WD, and Austin RH. 2000. Long-lived amide I vibrational modes in myoglobin. Phys. Rev. Lett. 84, 5435-5438.

21)@ Hoff WD, Jung K, Spudich JL. 1997. Molecular aspects of photosignaling by archaeal sensory rhodopsins. Ann. Rev. Biophys. Biomol. Struct. 26, 223-258.

22)@ Kort R, Vonk H, Xu X, Hoff WD, Crielaard W, Hellingwerf KJ. 1996. Evidence for the trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein. FEBS Lett. 382, 73-78.

23)@ Xie A, Hoff WD, Kroon AR, Hellingwerf KJ. 1996. Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein. Biochemistry 35, 14671-14678.

24)@ Hoff WD, Düx P, Hård K, Devreese B, Nugteren-Roodzant IM, Crielaard W, Boelens, R, van Beeumen J, Hellingwerf KJ. 1994. Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry. Biochemistry 33, 13959-13962.

25)@ Hoff WD, van Stokkum IHM, van Ramesdonk HJ, van Brederode ME, Brouwer AM, Fitch JC, Meyer TE, van Grondelle R, Hellingwerf KJ. 1994. Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila. Biophys. J. 67, 1691-1705.

26)@ Sprenger WW, Hoff WD, Armitage JP, Hellingwerf KJ. 1993. The Eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorbance spectrum of the photoactive yellow protein. J. Bacteriol. 175, 3096-3104.