Aihua Xie

Identification of Six New Photoactive Yellow Proteins—Diversity and Structure–Function Relationships in a Bacterial Blue Light Photoreceptor†

Photoactive yellow proteins (PYP) are bacterial photoreceptors with a Per‐Arnt‐Sim (PAS) domain fold. We report the identification of six new PYPs, thus nearly doubling the size of this protein family. This extends the taxonomic diversity of PYP‐containing bacteria from photosynthetic to nonphotosynthetic bacteria, from aquatic to soil‐dwelling organisms, and from Proteobacteria to Salinibacter ruber from the phylum Bacteriodetes. The new PYPs greatly increase the sequence diversity of the PYP family, reducing the most prevalent pair‐wise identity from 45% to 25%. Sequence alignments and analysis indicate that all 14 PYPs share a common structure with 13 highly conserved residues that form the chromophore binding pocket. Nevertheless, the functional properties of the PYPs vary greatly—the absorbance maximum extends from 432 to 465 nm, the pKa of the chromophore varies from pH 2.8 to 10.2, and the lifetime of the presumed PYP signaling state ranges from 1 ms to 1 h. Thus, the PYP family offers an excellent opportunity to investigate how functional properties are tuned over a wide range, while maintaining the same overall protein structural fold. We discuss the implications of these results for structure–function relationships in the PYP family.

Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis

Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with photochemistry based on trans to cis isomerization of its p-coumaric acid (pCA) chromophore. The addition of trans-locked pCA to I. loihiensis increases biofilm formation, whereas cis-locked pCA decreases it. This demonstrates that the PYP homologue regulates biofilm formation in I. loihiensis, revealing an unexpected functional versatility in the PYP family of photoreceptors. These results imply that I. loihiensis thrives not only in the deep sea but also near the water surface and provide an example of genome-based discovery of photophysiological responses. The use of locked pCA analogs is a novel and generally applicable pharmacochemical tool to study the in vivo role of PYPs irrespective of genetic accessibility. Heterologously produced PYP from I. loihiensis (Il PYP) absorbs maximally at 446 nm and has a pCA pK(a) of 3.4. Photoexcitation triggers the formation of a pB signaling state that decays with a time constant of 0.3 s. FTIR difference signals at 1726 and 1497 cm(-1) reveal that active-site proton transfer during the photocycle is conserved in Il PYP. It has been proposed that a correlation exists between the lifetime of a photoreceptor signaling state and the time scale of the biological response that it regulates. The data presented here provide an example of a protein with a rapid photocycle that regulates a slow biological response.

Strong Ionic Hydrogen Bonding Causes a Spectral Isotope Effect in Photoactive Yellow Protein

Standard hydrogen bonds are of great importance for protein structure and function. Ionic hydrogen bonds often are significantly stronger than standard hydrogen bonds and exhibit unique properties, but their role in proteins is not well understood. We report that hydrogen/deuterium exchange causes a redshift in the visible absorbance spectrum of photoactive yellow protein (PYP). We expand the range of interpretable isotope effects by assigning this spectral isotope effect (SIE) to a functionally important hydrogen bond at the active site of PYP. The inverted sign and extent of this SIE is explained by the ionic nature and strength of this hydrogen bond. These results show the relevance of ionic hydrogen bonding for protein active sites, and reveal that the inverted SIE is a novel, to our knowledge, tool to probe ionic hydrogen bonds. Our results support a classification of hydrogen bonds that distinguishes the properties of ionic hydrogen bonds from those of both standard and low barrier hydrogen bonds, and show how this classification helps resolve a recent debate regarding active site hydrogen bonding in PYP.

Side-chain specific isotopic labeling of proteins for infrared structural biology: The case of ring-D4-tyrosine isotope labeling of photoactive yellow protein

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling.

A Conserved Helical Capping Hydrogen Bond in PAS Domains Controls Signaling Kinetics in the Superfamily Prototype Photoactive Yellow Protein

PAS domains form a divergent protein superfamily with more than 20 000 members that perform a wide array of sensing and regulatory functions in all three domains of life. Only nine residues are well-conserved in PAS domains, with an Asn residue at the start of α-helix 3 showing the strongest conservation. The molecular functions of these nine conserved residues are unknown. We use static and time-resolved visible and FTIR spectroscopy to investigate receptor activation in the photosensor photoactive yellow protein (PYP), a PAS domain prototype. The N43A and N43S mutants allow an investigation of the role of side-chain hydrogen bonding at this conserved position. The mutants exhibit a blue-shifted visible absorbance maximum and up-shifted chromophore pK(a). Disruption of the hydrogen bonds in N43A PYP causes both a reduction in protein stability and a 3400-fold increase in the lifetime of the signaling state of this photoreceptor. A significant part of this increase in lifetime can be attributed to the helical capping interaction of Asn43. This extends the known importance of helical capping for protein structure to regulating functional protein kinetics. A model for PYP activation has been proposed in which side-chain hydrogen bonding of Asn43 is critical for relaying light-induced conformational changes. However, FTIR spectroscopy shows that both Asn43 mutants retain full allosteric transmission of structural changes. Analysis of 30 available high-resolution structures of PAS domains reveals that the side-chain hydrogen bonding of residue 43 but not residue identity is highly conserved and suggests that its helical cap affects signaling kinetics in other PAS domains.

Photoreceptors Take Charge: Emerging Principles for Light Sensing

The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation.Wediscuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes. Expected final online publication date for the Annual Review of Biophysics Volume 47 is May 20, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Long-lived amide I vibrational modes in myoglobin: breathers in biology

We show that myoglobin, which is almost entirely α helix in secondary structure, has an unusually long-lived 12 ps vibrational excited state lifetime generated by optically pumping at the blue side 5.85 microns of the amide I band, indicating the generation of a long-lived trapped soliton- antisoliton breather mode.

Impact of Hofmeister Salts on Structural Dynamics of Photoactive Yellow Protein

Water is known as the lubricant of life. Without water, most proteins would lose their biological activities. Extensive studies have been carried out on how aqueous solutions with high concentration salts alter the stability and solubility of proteins. Such effects are thought to be mediated largely via salt-water interactions and water-protein interactions. This classic research field is known as the Hofmeister Series. We report the effects of Hofmeister salts on the structural dynamics of proteins. Photoactive yellow protein (PYP), a bacterial blue light photoreceptor protein, is employed as a model system in this study. Time-resolved FTIR spectroscopic techniques were used to probe the protein structural changes of PYP in response to blue light excitation. Our data demonstrate that high concentration salt solutions have profound effects on functionally important motions of PYP, including (1) the light triggered proton transfer pathway in the active site and (2) the large conformational changes associated with PYP receptor activation. We will discuss the significance of our study in relation with protein crystallization, and with other properties of the Hofmeister series.

Impacts of Crystallization on Protein Structural Dynamics

X‐ray crystallographic technique is a powerful technique for structural determination of steady state proteins. Now this technique has been further developed to determine the structures of transient states of proteins during their functional processes. Can a protein function in the crystalline state? It is often assumed but untested that the functionally important structural dynamics of a protein are preserved in the crystalline state. This lack of test is largely due to the fact that major structural determination techniques, x‐ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, can only be applied to either crystalline state or solution state, not both. Here we report our direct study on the impacts of protein crystallization on the structural dynamics of a blue light photoreceptor protein using time‐resolved Fourier transform infrared (FTIR) difference spectroscopic techniques. We found that proteins in crystalline state experience suppressed conformational changes, accelerated kinet...