Nikita Rozanov

I am an Oregon State Chemical Engineering and Physics graduate. My interests are in using software and machine learning to make an impact. After graduation I worked at Intel Corporation in the manufacturing research and development department for three years. Recently, I joined Amazon.com as a System Development Engineer, focusing on network automation. I am excited to showcase the research and projects from my undergraduate years and beyond. I also include some of my artwork on my website.

selected publications

Rational design for high bioorthogonal fluorogenicity of tetrazine-encoded green fluorescent proteins

Longteng Tang, Riley M. Bednar, Nikita D. Rozanov, Marcus L. Hemshorn, Ryan A. Mehl, and Chong Fang

Natural Sciences 2022

Abs Link

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine-encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability but suffer from low fluorogenicity. Here, we unveil the real-time fluorescence mechanisms by investigating two site-specific tetrazine-modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150-Tet with a fluorescence turn-on ratio of ∼9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet-v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn-on ratios to ∼14/31, making the fluorogenicity of GFP150-Tet-S202Y the highest among all up-to-date tetrazine-encoded GFPs. In live eukaryotic cells, the GFP150-Tet-v3.0-S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy.
Switching between ultrafast pathways enables a green-red emission ratiometric fluorescent-protein-based Ca²⁺ biosensor

Longteng Tang, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell, and Chong Fang

International Journal of Molecular Sciences 2021

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Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca²⁺ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca²⁺-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca²⁺ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca²⁺ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (∆R/R₀) of 300%, outperforming many FRET-and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.
Dissecting optical response and molecular structure of fluorescent proteins with non-canonical chromophores

Breland G. Oscar, Liangdong Zhu, Hayati Wolfendeen, Nikita D. Rozanov, Alvin Chang, Kenneth T. Stout, Jason W. Sandwisch, Joseph J. Porter, Ryan A. Mehl, and Chong Fang

Frontiers in Molecular Biosciences 2020

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Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the ortho site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of 4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a 30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm⁻¹ in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.
Surface coating structure and its interaction with cytochrome c in EG6-coated nanoparticles varies with surface curvature

Clyde A. Daly, Caley Allen, Nikita D. Rozanov, Gene Chong, Eric S. Melby, Thomas R. Kuech, Samuel E. Lohse, Catherine J. Murphy, Joel A. Pedersen, and Rigoberto Hernandez

Langmuir 2020

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The composition, orientation, and conformation of proteins in biomolecular coronas acquired by nanoparticles in biological media contribute to how they are identified by a cell. While numerous studies have investigated protein composition in biomolecular coronas, relatively little detail is known about how the nanoparticle surface influences the orientation and conformation of the proteins associated with them. We previously showed that the peripheral membrane protein cytochrome c adopts preferred poses relative to negatively charged 3-mercaptopropionic acid (MPA)-gold nanoparticles (AuNPs). Here, we employ molecular dynamics simulations and complementary experiments to establish that cytochrome c also assumes preferred poses upon association with nanoparticles functionalized with an uncharged ligand, specifically ω-(1-mercaptounde-11-cyl)hexa(ethylene glycol) (EG6). We find that the display of the EG6 ligands is sensitive to the curvature of the surface - and, consequently, the effective diameter of the nearly spherical nanoparticle core - which in turn affects the preferred poses of cytochrome c.