Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications

β‑Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields

A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM β-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP–PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site. Conversely, the same linkage in the descendant TEM–PenG experiences large electric fields that stabilize the more charge-separated transition state and thus lower the free energy barrier to hydrolysis. Specifically, the electric fields were improved from −59 to −140 MV/cm in an ancient evolution dating back billions of years, contributing 5 orders of magnitude rate acceleration. This trend continues today in the nullification of newly developed antibiotic drugs. The fast linkage hydrolysis acquired from evolution is counteracted by the upgrade of PenG to Avb whose linkage escapes from the hydrolysis by returning to a low-field environment. Using the framework of electrostatic catalysis, the electric field, an observable from vibrational spectroscopy, provides a unifying physical metric to understand protein evolution and to guide the design of covalent drugs

GFP Variants with Alternative β‑Strands and Their Application as Light-driven Protease Sensors: A Tale of Two Tails

Green fluorescent protein (GFP) variants that carry one extra strand 10 (s10) were created and characterized, and their possible applications were explored. These proteins can fold with either one or the other s10, and the ratio of the two folded forms, unambiguously distinguished by their resulting colors, can be systematically modulated by mutating the residues on s10 or by changing the lengths of the two inserted linker sequences that connect each s10 to the rest of the protein. We have discovered robust empirical rules that accurately predict the product ratios of any given construct in both bacterial and mammalian expressions. Exploiting earlier studies on photodissociation of cut s10 from GFP (Do and Boxer, 2011), ratiometric protease sensors were designed from the construct by engineering a specific protease cleavage site into one of the inserted loops, where the bound s10 is replaced by the other strand upon protease cleavage and irradiation with light to switch its color. Since the conversion involves a large spectral shift, these genetically encoded sensors display a very high dynamic range. Further engineering of this class of proteins guided by mechanistic understanding of the light-driven process will enable interesting and useful application of the protein

Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications

Structural Insight into the Photochemistry of Split Green Fluorescent Proteins: A Unique Role for a His-Tag

Oligohistidine affinity tags (His-tags) are commonly fused to proteins to aid in their purification via metal affinity chromatography. These His-tags are generally assumed to have minimal impact on the properties of the fusion protein, as they have no propensity to form ordered elements, and are small enough not to significantly affect the solubility or size. Here we report structures of two variants of truncated green fluorescent protein (GFP), i.e., split GFP with a β-strand removed, that were found to behave differently in the presence of light. In these structures, the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides an explanation for the seemingly very different peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications

A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

The vibrational Stark effect (VSE) has been used to measure the electric field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with Δ<i>G</i>^⧧ in a series of conventional mutants, yielding an estimate for the electrostatic contribution to catalysis (Fried et al. <i>Science</i> <b>2014</b>, <i>346</i>, 1510–1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The electric fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is observed between the electric field and Δ<i>G</i>^⧧ that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and calculation are developed to estimate changes in the electric field accompanying changes in the extended hydrogen-bond network in the active site. The results are consistent with a model in which the O–H group of a key active site tyrosine functions by imposing a static electrostatic potential onto the carbonyl bond. The model suggests that the contribution to catalysis from the active site hydrogen bonds is of similar weight to the distal interactions from the rest of the protein. A similar linear correlation was also observed between the proton affinity of KSI’s active site and the catalytic rate, suggesting a direct connection between the strength of the H-bond and the electric field it exerts

Thermodynamics, Kinetics, and Photochemistry of β-Strand Association and Dissociation in a Split-GFP System

Truncated green fluorescent protein (GFP) that is refolded after removing the 10th β-strand can readily bind to a synthetic strand to recover the absorbance and fluorescence of the whole protein. This allows rigorous experimental determination of thermodynamic and kinetic parameters of the split system including the equilibrium constant and the association/dissociation rates, which enables residue-specific analysis of peptide–protein interactions. The dissociation rate of the noncovalently bound strand is observed by strand exchange that is accompanied by a color change, and surprisingly, the rate is greatly enhanced by light irradiation. This peptide–protein photodissociation is a very unusual phenomenon and can potentially be useful for introducing spatially and temporally well-defined perturbations to biological systems as a genetically encoded caged protein

Genetic Code Expansion in <i>Rhodobacter sphaeroides</i> to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers

Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for <i>E. coli</i> into the model bacterium needed to produce RCs, <i>Rhodobacter sphaeroides</i>. Plasmids were developed and optimized to incorporate 3-chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in <i>R. sphaeroides</i>, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from <i>E. coli</i>. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein <i>via</i> mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in <i>R. sphaeroides</i> expands research capabilities in the photosynthetic field

Atomic Recombination in Dynamic Secondary Ion Mass Spectrometry Probes Distance in Lipid Assemblies: A Nanometer Chemical Ruler

The lateral organization of biological membranes is thought to take place on the nanometer length scale. However, this length scale and the dynamic nature of small lipid and protein domains have made characterization of such organization in biological membranes and model systems difficult. Here we introduce a new method for measuring the colocalization of lipids in monolayers and bilayers using stable isotope labeling. We take advantage of a process that occurs in dynamic SIMS called atomic recombination, in which atoms on different molecules combine to form diatomic ions that are detected with a NanoSIMS instrument. This process is highly sensitive to the distance between molecules. By measuring the efficiency of the formation of ¹³C¹⁵N^– ions from ¹³C and ¹⁵N atoms on different lipid molecules, we measure variations in the lateral organization of bilayers even though these heterogeneities occur on a length scale of only a few nm, well below the diameter of the primary ion beam of the NanoSIMS instrument or even the best super-resolution fluorescence methods. Using this technique, we provide direct evidence for nanoscale phase separation in a model membrane, which may provide a better model for the organization of biological membranes than lipid mixtures with microscale phase separation. We expect this technique to be broadly applicable to any assembly where very short scale proximity is of interest or unknown, both in chemical and biological systems

Short Hydrogen Bonds and Proton Delocalization in Green Fluorescent Protein (GFP)

Short hydrogen bonds and specifically low-barrier hydrogen bonds (LBHBs) have been the focus of much attention and controversy for their possible role in enzymatic catalysis. The green fluorescent protein (GFP) mutant S65T, H148D has been found to form a very short hydrogen bond between Asp148 and the chromophore resulting in significant spectral perturbations. Leveraging the unique autocatalytically formed chromophore and its sensitivity to this interaction we explore the consequences of proton affinity matching across this putative LBHB. Through the use of noncanonical amino acids introduced through nonsense suppression or global incorporation, we systematically modify the acidity of the GFP chromophore with halogen substituents. X-ray crystal structures indicated that the length of the interaction with Asp148 is unchanged at ∼2.45 Å while the absorbance spectra demonstrate an unprecedented degree of color tuning with increasing acidity. We utilized spectral isotope effects, isotope fractionation factors, and a simple 1D model of the hydrogen bond coordinate in order to gain insight into the potential energy surface and particularly the role that proton delocalization may play in this putative short hydrogen bond. The data and model suggest that even with the short donor–acceptor distance (∼2.45 Å) and near perfect affinity matching there is not a LBHB, that is, the barrier to proton transfer exceeds the H zero-point energy