Regulation of Absorption and Emission in a Protein/Fluorophore Complex.

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission.

Light controlled reversible Michael addition of cysteine: a new tool for dynamic site-specific labeling of proteins.

Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex.

Design of Large Stokes Shift Fluorescent Proteins Based on Excited State Proton Transfer of an Engineered Photobase.

The incredible potential for fluorescent proteins to revolutionize biology has inspired the development of a variety of design strategies to address an equally broad range of photophysical characteristics, depending on potential applications. Of these, fluorescent proteins that simultaneously exhibit high quantum yield, red-shifted emission, and wide separation between excitation and emission wavelengths (Large Stokes Shift, LSS) are rare. The pursuit of LSS systems has led to the formation of a complex, obtained from the marriage of a rationally engineered protein (human cellular retinol binding protein II, hCRBPII) and different fluorogenic molecules, capable of supporting photobase activity.

Human Cellular Retinol Binding Protein II Forms a Domain-Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering.

Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein-engineering strategy for tailoring functional diversity. However, domain swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member hCRBPII, and suggest a mechanism for domain-swapped trimerization.

FGF-2 and HGF reverse abiraterone's effect οn intracellular levels of DHT in androgen-dependent and androgen independent prostate cancer cell lines.

Purpose: Growth factors such as fibroblast growth factor 2 (FGF-2) and hepatocyte growth factor (HGF) appear at high levels in prostate cancer (PC). Abiraterone is an androgen biosynthesis inhibitor which is currently in use as a standard treatment in clinics to impair tumor growth. Development of resistance to anticancer therapies is unfortunately a very common feature of cancer cells that threatens the patient lives.

Engineering the hCRBPII Domain-Swapped Dimer into a New Class of Protein Switches.

Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain-swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches.

Engineering of a Red Fluorogenic Protein/Merocyanine Complex for Live-Cell Imaging.

A reengineered human cellular retinol binding protein II (hCRBPII), a 15-kDa protein belonging to the intracellular lipid binding protein (iLBP) family, generates a highly fluorescent red pigment through the covalent linkage of a merocyanine aldehyde to an active site lysine residue. The complex exhibits "turn-on" fluorescence, due to a weakly fluorescent aldehyde that "lights up" with subsequent formation of a strongly fluorescent merocyanine dye within the binding pocket of the protein. Cellular penetration of merocyanine is rapid, and fluorophore maturation is nearly instantaneous.

Mimicking Microbial Rhodopsin Isomerization in a Single Crystal.

Bacteriorhodopsin represents the simplest, and possibly most abundant, phototropic system requiring only a retinal-bound transmembrane protein to convert photons of light to an energy-generating proton gradient. The creation and interrogation of a microbial rhodopsin mimic, based on an orthogonal protein system, would illuminate the design elements required to generate new photoactive proteins with novel function. We describe a microbial rhodopsin mimic, created using a small soluble protein as a template, that specifically photoisomerizes all- trans to 13- cis retinal followed by thermal relaxation to the all- trans isomer, mimicking the bacteriorhodopsin photocycle, in a single crystal.

A Near-Infrared Photoswitchable Protein-Fluorophore Tag for No-Wash Live Cell Imaging.

FR-1V, a fluorene-based aldehydic chromophore, binds its target protein as an imine to yield a highly bathochromic pigment, CF-2, a prototypic protein-dye tagging system whose NIR emission can be spatiotemporally switched ON by rapid UV-light activation. This is achieved through photoisomerization of the imine and its subsequent protonation. We demonstrate a no-wash protocol for live cell imaging of subcellular compartments in a variety of mammalian cell lines with minimal fluorescence background.

A Genetically Encoded Ratiometric pH Probe: Wavelength Regulation-Inspired Design of pH Indicators.

Mutants of human cellular retinol-binding protein II (hCRBPII) were engineered to bind a julolidine retinal analogue for the purpose of developing a ratiometric pH sensor. The design relied on the electrostatic influence of a titratable amino acid side chain, which affects the absorption and, thus, the emission of the protein/fluorophore complex. The ratio of emissions obtained at two excitation wavelengths that correspond to the absorption of the two forms of the protein/fluorophore complex, leads to a concentration-independent measure of pH.

Free-Energy-Based Protein Design: Re-Engineering Cellular Retinoic Acid Binding Protein II Assisted by the Moveable-Type Approach.

How to fine-tune the binding free energy of a small-molecule to a receptor site by altering the amino acid residue composition is a key question in protein engineering. Indeed, the ultimate solution to this problem, to chemical accuracy (±1 kcal/mol), will result in profound and wide-ranging applications in protein design. Numerous tools have been developed to address this question using knowledge-based models to more computationally intensive molecular dynamics simulations-based free energy calculations, but while some success has been achieved there remains room for improvement in terms of overall accuracy and in the speed of the methodology.

Computationally Aided Absolute Stereochemical Determination of Enantioenriched Amines.

A simple and efficient protocol for sensing the absolute stereochemistry and enantiomeric excess of chiral monoamines is reported. Preparation of the sample requires a single-step reaction of the 1,1'-(bromomethylene)dinaphthalene (BDN) with the chiral amine. Analysis of the exciton coupled circular dichroism generated from the BDN-derivatized chiral amine sample, along with comparison to conformational analysis performed computationally, yields the absolute stereochemistry of the parent chiral monoamine.

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein.

A Photoisomerizing Rhodopsin Mimic Observed at Atomic Resolution.

The members of the rhodopsin family of proteins are involved in many essential light-dependent processes in biology. Specific photoisomerization of the protein-bound retinylidene PSB at a specified wavelength range of light is at the heart of all of these systems. Nonetheless, it has been difficult to reproduce in an engineered system.

Host-Guest Assembly of a Molecular Reporter with Chiral Cyanohydrins for Assignment of Absolute Stereochemistry.

The absolute stereochemistry of cyanohydrins, derived from ketones and aldehydes, is obtained routinely, in a microscale and derivatization-free manner, upon their complexation with Zn-MAPOL, a zincated porphyrin host with a binding pocket comprised of a biphenol core. The host-guest complex leads to observable exciton-coupled circular dichroism (ECCD), the sign of which is easily correlated to the absolute stereochemistry of the bound cyanohydrin. A working model, based on the ECCD signal of cyanohydrins with known configuration, is proposed.

Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch.

Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa , leading to protonation of the imine and subsequent "turn-on" of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa , leading to loss of color because the imine is not protonated.

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2-11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation.

Structures of holo wild-type human cellular retinol-binding protein II (hCRBPII) bound to retinol and retinal.

Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket.

Rational design of a colorimetric pH sensor from a soluble retinoic acid chaperone.

Reengineering of cellular retinoic acid binding protein II (CRABPII) to be capable of binding retinal as a protonated Schiff base is described. Through rational alterations of the binding pocket, electrostatic perturbations of the embedded retinylidene chromophore that favor delocalization of the iminium charge lead to exquisite control in the regulation of chromophoric absorption properties, spanning the visible spectrum (474-640 nm). The pKa of the retinylidene protonated Schiff base was modulated from 2.

Toward an understanding of the retinal chromophore in rhodopsin mimics.

Recently, a rhodopsin protein mimic was constructed by combining mutants of the cellular retinoic acid binding protein II (CRABPII) with an all-trans retinal chromophore. Here, we present a combined computational quantum mechanics/molecular mechanics (QM/MM) and experimental ultrafast kinetic study of CRABPII. We employ the QM/MM models to study the absorption (λ(a)max), fluorescence (λ(f)max), and reactivity of a CRABPII triple mutant incorporating the all-trans protonated chromophore (PSB-KLE-CRABPII).

Tuning the electronic absorption of protein-embedded all-trans-retinal.

Protein-chromophore interactions are a central component of a wide variety of critical biological processes such as color vision and photosynthesis. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside the protein cavity, we redesigned human cellular retinol binding protein II (hCRBPII) to fully encapsulate all-trans-retinal and form a covalent bond as a protonated Schiff base. This system, using rational mutagenesis designed to alter the electrostatic environment within the binding pocket of the host protein, enabled regulation of the absorption maximum of the pigment in the range of 425 to 644 nanometers.

A minimalist NMR approach for the structural revision of mucoxin.

In an attempt to revise the structural assignment of mucoxin, and faced with 64 diastereomeric possibilities, we resorted to the synthesis of truncated structures that contained the core stereochemical sites. Twelve stereochemical analogues were synthesized, their (1)H and (13)C NMR spectra were analyzed and four recurring stereochemical trends were distilled from the data. Applying the observed trends to the diastereomeric population pared the possible choices for the correct structure of mucoxin from 64 to 4.

Magnetic glyco-nanoparticles: a tool to detect, differentiate, and unlock the glyco-codes of cancer via magnetic resonance imaging.

Within cancer, there is a large wealth of diversity, complexity, and information that nature has engineered rendering it challenging to identify reliable detection methods. Therefore, the development of simple and effective techniques to delineate the fine characteristics of cancer cells can have great potential impacts on cancer diagnosis and treatment. Herein, we report a magnetic glyco-nanoparticle (MGNP) based nanosensor system bearing carbohydrates as the ligands, not only to detect and differentiate cancer cells but also to quantitatively profile their carbohydrate binding abilities by magnetic resonance imaging (MRI).

Elucidating the exact role of engineered CRABPII residues for the formation of a retinal protonated Schiff base.

Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered to specifically bind and react with all-trans-retinal to form a protonated Schiff base. Each step of this process has been dissected and four residues (Lys132, Tyr134, Arg111, and Glu121) within the CRABPII binding site have been identified as crucial for imine formation and/or protonation. The precise role of each residue has been examined through site directed mutagenesis and crystallographic studies.

Dissection of the critical binding determinants of cellular retinoic acid binding protein II by mutagenesis and fluorescence binding assay.

The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by providing a carboxylic acid dimer partner in the form of a Glu residue.