Scientific Contribution

Unraveling Cryptochrome's Light-Sensing Mechanism (Ph.D.)

Circadian rhythms, the innate clocks governing our body's functions in tune with the day-night cycles, are sensitive to changes in environmental conditions such as light and temperature. In the widely-researched biological system, fruit fly, circadian rhythms are regulated through an intricate dance between specific proteins, namely Timeless (TIM) and Period (PER). These proteins establish a cyclical pattern that drives many physiological processes. An additional player, Cryptochrome (CRY), brings an extra layer of complexity to this system. It fine-tunes the rhythms to align with light. It does so by steering TIM towards the path of degradation facilitated by a proteasome system. However, the mechanism of how CRY responds to light remains a subject of ongoing discussions, particularly concerning the question of whether the light-dependent reduction of its cofactor, flavin adenine dinucleotide (FAD), is related to alterations in CRY's structure, including the undocking of the C-terminus tail (CTT).

In pursuit of answers to this debate, I delved into the details of the FAD photoreduction process. I brought to light the crucial role played by a chain of four tryptophan residues in the electron transfer, which is central to FAD reduction. Manipulating these residues by either substitution or repositioning led to changes in the FAD reduction process. Importantly, such manipulations resulted in discernible effects on the conformational activation of CRY and its biological interplay with TIM.

This study illuminates the long-standing question surrounding CRY's light response mechanism and its relationship with FAD reduction. It marks a step towards understanding proteins that underpin the circadian rhythms in fruit flies. Moreover, this research programmed CRY variants with different light sensitivities, thereby expanding the potential scope and application of optogenetics.

a.     Lin C, Top D, Manahan CC, Young MW, Crane BR. Circadian clock activity of cryptochrome relies on tryptophan-mediated photoreduction. Proc Natl Acad Sci U S A. 2018 Apr 10;115(15):3822-3827. PubMed Central PMCID: PMC5899454. 

b.     Chandrasekaran S, Schneps CM, Dunleavy R, Lin C, DeOliveira CC, Ganguly A, Crane BR. Tuning flavin environment to detect and control light-induced conformational switching in Drosophila cryptochrome. Commun Biol. 2021 Feb 26;4(1):249. PubMed Central PMCID: PMC7910608. 

c.     Yee EF, Chandrasekaran S, Lin C, Crane BR. Physical methods for studying flavoprotein photoreceptors. Methods Enzymol. 2019;620:509-544. PubMed Central PMCID: PMC6512857. 

d.     Foroutannejad, S., Good, L.L., Lin, C., Carter, Z.I., Tadesse, M.G., Lucius, A.L., Crane, B.R., and Maillard, R.A. (2023). The cofactor-dependent folding mechanism of Drosophila cryptochrome revealed by single-molecule pulling experiments. Nat Commun 14, 1057. 10.1038/s41467-023-36701-y.

Decoding Drosophila's Light-Responding Module: The CRY-TIM Complex (Ph.D.)

The circadian clock of Drosophila melanogaster operates via an intricate mechanism, at the heart of which lies the light-responsive CRY-TIM complex. Understanding this complex is critical to unlocking the secrets of the circadian clock's resetting mechanism. However, studying this dynamic has presented unique challenges, primarily due to the large unstable protein TIM characterized by intrinsically unstructured regions. As a co-author in 2016, I contributed to work that utilized molecular dynamic (MD) simulations, identifying residue His378 as the crucial bridge connecting the redox state of FAD and the conformation of CRY's C-terminal tail (CTT). However, we were unable to directly monitor the heterodimerization of CRY and TIM as an indicator of CTT dynamics.

To overcome this limitation, I developed a new assay, independent of western-blot, that uses fluorescent detection for the rapid and efficient monitoring of the light-dependent interaction between CRY and TIM. This led to the discovery of the involvement of residue His377 in the gating mechanism of the C-terminal tail, a previously unreported finding. We also successfully reconstituted the CRY-TIM complex using transient expression in S2 insect cells and a nanobody-based affinity tag. With the aid of cross-linking mass spectrometry (XL-MS) and cryo-electron microscopy (cryo-EM), we were able to reveal the interfacing residues and a 3D structural model of this elusive heterodimer.

Our study led to several discoveries. We highlighted the N-terminal-dependent binding mechanism of TIM to CRY and illuminated the role of different TIM variants in the fine-tuning of the fruit fly body clock and environmental adaptation. We identified a "groove" region on TIM, crucial for its entry into the cell nucleus. Finally, we discovered the functional role of CRY's C-terminal tail in competing with TIM for binding sites, a mechanism that allows TIM binding exclusively under light conditions. These findings not only enhance our understanding of the core complex of the Drosophila circadian clock, but they also pave the way for the engineering of optogenetics protein pairs for various applications. 

a.     Lin, C., S. Feng, C. C. DeOliveira and B. R. Crane (2023). "Cryptochrome-Timeless structure reveals circadian clock timing mechanisms." Nature 617(7959): 194-199.

b.     Lin, C., C. M. Schneps, S. Chandrasekaran, A. Ganguly and B. R. Crane (2022). "Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome." Structure 30(6): 851-861 e855.

c.     Ganguly A, Manahan CC, Top D, Yee EF, Lin C, Young MW, Thiel W, Crane BR. Changes in active site histidine hydrogen bonding trigger cryptochrome activation. Proc Natl Acad Sci U S A. 2016 Sep 6;113(36):10073-8. PubMed Central PMCID: PMC5018803. 

Human Blood-Brain Barrier Receptor-Guided Evolution of AAVs for Brain Gene Therapy (Postdoc)

While significant advances in CRISPR and Prime Editing have been made, effective delivery of gene therapies to target brain cells remains challenging. My research focuses on developing novel delivery tools, specifically harnessing Adeno-associated viral vectors (AAVs). These non-pathogenic vectors have the natural  ability to cross the blood-brain barrier (BBB). However, their natural serotypes often affect other organs like the liver, thus necessitating the engineering of AAVs for more precise brain-targeted therapies.

To date, many engineered AAVs have shown promise in mice and non-human primates, but none have achieved optimal performance in humans. One reason for this limitation is the traditional method of directed evolution used in the development of AAVs in animals. This process involves infecting the animals with a library of AAV variants, followed by isolating those that prominently reach and enrich in the brain. This approach is resource-heavy and has limited success due to receptor differences between humans and other animals. 

In response, we've developed an in vitro strategy focused on evolving AAVs to target human BBB receptors, which is primarily concentrated in the primate brain, unlike other universally available transporters, which suggests AAVs targeting this receptor may have fewer off-target effects. Utilizing a cell-free resin-based selection coupled with cell-based infectivity tests, we efficiently isolated AAVs that bind to human receptor. Our approach was validated using known receptor-binder pairs targeting LY6A and msCar4.