Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. RFs have structural motifs that recognize stop codons in the decoding center and a GGQ motif for induction of hydrolysis in the peptidyl transfer center 70 Å away. Surprisingly, free RF2 is compact, with only 20 Å between its codon-reading and GGQ motifs. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Here we use time-resolved cryo-EM to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. About 25% of complexes have RFs in the compact state at 24 ms reaction time, and within 60 ms virtually all ribosome-bound RFs are transformed to their extended forms.

Late steps in bacterial translation initiation visualized using time-resolved cryo-EM.

The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2-5 and 70S4,6-8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip.

With the advent of direct electron detectors, cryo-EM has become a popular choice for molecular structure determination. Among its advantages over X-ray crystallography are (1) no need for crystals, (2) much smaller sample volumes, and (3) the ability to determine multiple structures or conformations coexisting in one sample. In principle, kinetic experiments can be done using standard cryo-EM, but mixing and freezing grids require several seconds. However, many biological processes are much faster than that time scale, and the ensuing short-lived states of the molecules cannot be captured. Here, we lay out a detailed protocol for how to capture such intermediate states on the millisecond time scale with time-resolved cryo-EM.

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM.

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms.

Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.

Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy.

Ribosomal subunit association is a key checkpoint in translation initiation but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding, and ribosome recycling, are amenable to study with this method.

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.

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.

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.