Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C‑Terminal Signaling in the Blue Light Sensor Plant Cryptochrome

Plant cryptochromes are photosensory receptors that regulate various central aspects of plant growth and development. These receptors consist of a photolyase homology region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a cryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV–vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid 396 to the neutral radical within 3.5 μs. The analysis revealed the existence of three intermediates characterized by changes in secondary structure. A marked loss of β-sheet structure is observed in the second intermediate evolving with a time constant of 500 μs. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV–vis. The only β-sheet in the PHR is located within the α/β subdomain, ∼25 Å away from the flavin. This subdomain has been previously attributed a role as a putative antenna binding site, but is now suggested to have evolved to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT

Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy

Cryptochromes act as blue light sensors in plants, insects, fungi, and bacteria. Recently, an animal-like cryptochrome (aCRY) was identified in the green alga <i>Chlamydomonas reinhardtii</i> by which gene expression is altered in response to not only blue light but also yellow and red light. This unique response of a flavoprotein <i>in vivo</i> has been attributed to the fact that the neutral radical of the flavin chromophore acts as dark form of the sensor, which absorbs in almost the entire visible spectral range (<680 nm). Here, we investigated light-induced processes in the protein moiety of full-length aCRY by UV–vis and Fourier transform infrared spectroscopy. Findings are compared to published results on the homologous (6-4) photolyases, DNA repair enzymes. The oxidized state of aCRY is converted to the neutral radical by blue light. The recovery is strongly dependent on pH and might be catalyzed by a conserved histidine of the (6-4)/clock cluster. The decay is independent of oxygen concentration in contrast to that of other cryptochromes and (6-4) photolyases. This blue light reaction of the oxidized flavin is not accompanied by any detectable changes in secondary structure, in agreement with a role <i>in vivo</i> of an unphysiological preactivation. In contrast, the conversion by red light of the neutral radical to the anionic fully reduced state proceeds with conformational changes in turn elements, which most probably constitute a part of the signaling process. These changes have not been detected in the corresponding transition of (6-4) photolyase, which points to a decisive difference between the sensor and the enzyme

Improved Precursor Characterization for Data-Dependent Mass Spectrometry

Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultrafast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (<i>m</i>/<i>z</i>) space, which ultimately inhibits the acquisition of an MS/MS event. Here, we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated <i>m</i>/<i>z</i> regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53 000 in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of prefractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments