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. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution.

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. The large increase in basicity upon photoexcitation leads to protonation of the fluorophore in the excited state, dramatically red-shifting its emission, leading to an LSS protein/fluorophore complex. Essential for selective photobase activity is the intimate involvement of the target protein structure and sequence that enables Excited State Proton Transfer (ESPT). The potential power and usefulness of the strategy was demonstrated in live cell imaging of human cell lines.

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. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, thus demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site through the introduction of a single mutation, taking advantage of its threefold symmetry. Together, these studies show how nature can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.

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. This study aimed to investigate whether FGF-2 and HGF act as a possible resistant mechanism to the abiraterone activity on the androgen synthesis pathway in PC. METHODS: The intracellular levels of 17-OH progesterone and dihydrotestosterone (DHT) were determined by enzyme immunoassays in cell lysates of LNCaP and PC3 PC cells upon co-treatment of cells with abiraterone and FGF-2 or HGF. RESULTS: Abiraterone treatment resulted in significant reduction in the intracellular levels of 17-OH progesterone and DHT in both LnCap and PC3 cells. FGF-2 and HGF were found to decrease the intracellular levels of 17-OH progesterone in both cell lines, whereas HGF alone was found to increase the intracellular levels of DHT only in PC3 cells. However, the simultaneous exposure of cells to abiraterone and FGF-2 or HGF was found to result in an increase in the intracellular levels of DHT, while it did not result in changes in the intracellular levels of 17-OH progesterone. CONCLUSION: These findings suggest that FGF-2 and HGF may act as an escape mechanism, aiding the development of resistance to abiraterone by restoring intra-tumoral androgen synthesis that may contribute to disease progression.

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. The hCRBPII/merocyanine complex displays high quantum yield, low cytotoxicity, specificity in labeling organelles, and compatibility in both cancer cell lines and yeast cells. The hCRBPII/merocyanine tag is brighter than most common red fluorescent proteins.

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. Atomic resolution structures of the apo- and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.

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. The key element for selective isomerization is a tuned steric interaction between the chromophore and protein, similar to that seen in the microbial rhodopsins. It is further demonstrated that a single mutation converts the system to a protein photoswitch without chromophore photoisomerization or conformational change.

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. Here we report a fast, knowledge-based movable-type (MT)-based approach to estimate the absolute and relative free energy of binding as influenced by mutations in a small-molecule binding site in a protein. We retrospectively validate our approach using mutagenesis data for retinoic acid binding to the Cellular Retinoic Acid Binding Protein II (CRABPII) system and then make prospective predictions that are borne out experimentally. The overall performance of our approach is supported by its success in identifying mutants that show high or even sub-nano-molar binding affinities of retinoic acid to the CRABPII system.

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. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.

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. We have developed rhodopsin mimics, using intracellular lipid binding protein family members as scaffolds, to study fundamental aspects of protein/chromophore interactions. Herein we describe a system that specifically isomerizes the retinylidene protonated Schiff base both thermally and photochemically. This isomerization has been characterized at atomic resolution by quantitatively interconverting the isomers in the crystal both thermally and photochemically. This event is accompanied by a large pKa change of the imine similar to the pKa changes observed in bacteriorhodopsin and visual opsins during isomerization.

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. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance.

"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. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

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. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.

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 2.4 to 8.1, giving rise to a set of proteins of varying colors and pH sensitivities. These proteins were used to demonstrate a concentration-independent, ratiometric pH sensor.

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). We also study the spectroscopy of the same mutant incorporating the unprotonated chromophore and of another double mutant incorporating the neutral unbound retinal molecule held inside the pocket. Finally, for PSB-KLE-CRABPII, stationary fluorescence spectroscopy and ultrafast transient absorption spectroscopy resolved two different evolving excited state populations which were computationally assigned to distinct locally excited and charge-transfer species. This last species is shown to evolve along reaction paths describing a facile isomerization of the biologically relevant 11-cis and 13-cis double bonds. This work represents a first exploratory attempt to model and study these artificial protein systems. It also indicates directions for improving the QM/MM models so that they could be more effectively used to assist the bottom-up design of genetically encodable probes and actuators employing the retinal chromophore.

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. With only nine point mutations, the hCRBPII mutants induced a systematic shift in the absorption profile of all-trans-retinal of more than 200 nanometers across the visible spectrum.