Marc Zimmer

The Role of the Protein Matrix in Green Fluorescent Protein Fluorescence

Abstract In the ground state of the highly conjugated green fluorescent protein (GFP), the chromophore should be planar. However, numerous crystal structures of GFP and GFP-like proteins have been reported with slightly twisted chromophores. We have previously shown that the protein cavity surrounding the chromophore in wild-type GFP is not complementary with a planar chromophore. This study shows that the crystal structure of wild-type GFP is not an anomaly: most of the GFP and GFP-like proteins in the protein databank have a protein matrix that is not complementary with a planar chromophore. When the π-conjugation across the ethylenic bridge of the chromophore is removed the protein matrix will significantly twist the freely rotating chromophore from the relatively planar structures found in the crystal structures. The possible consequences of this nonplanar deformation on the photophysics of GFP are discussed. A volume analysis of the cis-trans-isomerization of HBDI, a GFP chromophore model compound, reveals that its hula-twist motion is volume conserving. This means that, if the GFP chromophore or GFP chromophore model compounds undergo a cis-trans-isomerization in a volume-constricting medium, such as a protein matrix or viscous liquid, it will probably isomerize by means of a HT–type motion.

Photophysics and Dihedral Freedom of the Chromophore in Yellow, Blue, and Green Fluorescent Protein

Green fluorescent protein (GFP) and GFP-like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore. According to quantum mechanical calculations, the excited state of chromophore model systems has significant dihedral freedom, which may lead to fluorescence quenching intersystem crossing. Molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green, yellow, and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state. Most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield. According to our calculations, the dihedral freedom of the systems studied (BFP > A5 > YFP > GFP) increases in the inverse order to the quantum yield. In all simulations, the chromophore undergoes a negatively correlated hula twist (also known as a bottom hula twist mechanism).

Photoisomerization of green fluorescent protein and the dimensions of the chromophore cavity

Abstract Green fluorescent protein (GFP) has a fairly large central cavity, which contains the chromophore, however it does not have a shape that is complementary with a planar chromophore. The protein exerts some strain on the chromophore when it is planar and the only reason planar chromophores are found in GFP is due to their delocalized π-electrons. Our calculations show that the protein environment of GFP allows the chromophore some rotational freedom, especially by a hula-twist and in the ϕ dihedral angle. The excited state, responsible for fluorescence, may therefore be twisted relative to the ground state. However, cis–trans photoisomerization cannot occur by a 180° rotation of the τ dihedral angle.

Conformational analysis of copper(II) 1,4,8,11-tetraazacyclotetradecane macrocyclic systems

Abstract A search of the Cambridge Structure Database for all copper (II) complexes with a 1,4,8,11-tetraazacyclotetradecane backbone was conducted. Eighty nine crystal structures were found which were analyzed using an agglomerative, hierarchical, single-link cluster analysis method. The cluster analysis effectively separated the different conformations and configurations of the macrocyclic complexes. Molecular mechanics was used to establish the effect of macrocyclic substituents on the conformations and configurations adopted by the copper(II) macrocyclic complexes.

Molecular Mechanics Evaluation of the Proposed Mechanisms for the Degradation of Urea by Urease

Abstract A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the NHis272-Ni1-O-Curea and Ni1-O-Curea-Nurea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional “His320 acts as a base” mechanism is unlikely, while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.

Conformational searching of transition metal compounds

To date, no conformational search of inorganic complexes has been reported that searches for all the conformations and configurations available to the complex. This is due to the various coordination geometries that transition metal ions can adopt and the difficulties in conducting conformational searches with systems that have connected ring systems, such as the ones formed when a metal ion binds a multidentate ligand. Using three test complexes {[Co(dien)2]3+, [Co(dien)(dpt)]3+, and [Co(hexamethylcyclam)(Cl)  2+ } the ability of the random kick (Cartesian stochastic Monte Carlo search) method and the Monte Carlo dihedral and positional method to find all conformations and geometric isomers was tested (dien, diethylenetriamine; dpt, di(3‐aminopropyl)amine; hexamethylcyclam: tet‐a, meso‐5,5,7,12,12,14‐hexamethyl‐1,4,8,11‐tetraazacyclotetradecane; tet‐b, racemic‐5,5,7,12,12,14‐hexamethyl‐1,4,8,11‐tetraazacyclotetradecane). Both methods are significant improvements on the current method by which all possible isomers are entered graphically and minimized individually. The major difficulty that was encountered was how to differentiate between the large number of similar conformations found. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1549–1558, 1999

Computational analysis of Thr203 isomerization in green fluorescent protein.

Green fluorescent protein (GFP) is an extensively used fluorescent tag. Photoisomerization between two spectroscopically distinct states in wild-type GFP is responsible for its two visible absorption bands at 398 nm (A) and 475 nm (B). We have used molecular mechanics and database analysis to support the suggestion of other researchers that the anionic form of the GFP chromophore is responsible for the B absorption band, while the phenol form is responsible for band A. The anionic (-Otyr, Nimid, Glu222H) species is the only form that has a low energy pathway allowing for isomerization of Thr203 to a conformation where it stabilizes the phenolate form and is therefore the most likely species responsible for the B absorption band. The rotation of the Thr203 side-chain is restricted; this may be significant in the formation of the intermediate state which is central to the photoisomerization. Our calculations support the most commonly accepted mechanism for photoisomerization, and we have shown that the 201LSTQS205 sequence does not allow a g+ conformation for Thr203.

A molecular mechanics and database analysis of the structural preorganization and activation of the chromophore-containing hexapeptide fragment in green fluorescent protein.

We propose that heterologous posttranslational chromophore formation in green fluorescent protein (GFP) occurs because the chromophore-forming amino acid residues 65SYG67 are preorganized and activated for imidazolinone ring formation. Based on extensive molecular mechanical conformational searching of the precursor hexapeptide fragment (64FSYGVQ69), we suggest that the presence of low energy conformations characterized by short contacts (approximately 3 A) between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 accounts for the initial step in posttranslational chromophore formation. Database searches showed that the tight turn required to establish the key short contact is a unique structural motif that is rarely found, except in other FSYG tetrapeptide sequences. Additionally, ab initio calculations demonstrated that an arginine side chain can hydrogen bond to the carbonyl oxygen of Ser65, activating this group for nucleophilic attack by the nearby lone pair of the Gly67 amide nitrogen. We propose that GFP chromophore-formation is initiated by a unique combination of conformational and electronic enhancements, identified by computational methods.

Empirical Force Field Analysis of the Revised Structure of Coenzyme F430. Epimerization and Geometry of the Corphinoid Tetrapyrrole

We undertook an empirical force field analysis of the conformational changes that accompany the diepimerization of coenzyme F430. The crystal structure of 12,13-diepi F430M was used as a test of the parameter set and as the basis for the calculations. The individual pyrrole rings in 13-epi and 12,13-diepi F430 adopt alternating half chair conformations leading to a ruffled macrocycle, native F430 is also ruffled but the individual pyrroles are planar. The 12,13 di-dehydro F430 and native F430 conformations are extremely similar, this accounts for the experimental observation that reduction of 12,13 di-dehydro-F430 forms native F430 and not 12,13-diepi F430. Native F430 can easily accommodate both square planar and, by bending, trigonal bipyramidal coordination geometries about nickel. We suggest that bent trigonal bipyramidal form is the conformer bound to the protein and that direct binding of the amino acid side chains to nickel is probably not important.

On the origin of fluorescence in bacteriophytochrome infrared fluorescent proteins

Tsien et al. (Science, 2009, 324, 804-807) recently reported the creation of the first infrared fluorescent protein (IFP). It was engineered from bacterial phytochrome by removing the PHY and histidine kinase-related domains, by optimizing the protein to prevent dimerization, and by limiting the biliverdins conformational freedom, especially around its D ring. We have used database analyses and molecular dynamics simulations with freely rotating chromophoric dihedrals in order to model the dihedral freedom available to the biliverdin D ring in the excited state and to show that the tetrapyrrole ligands in phytochromes are flexible and can adopt many conformations; however, their conformational space is limited/defined by the chemospatial characteristics of the protein cavity. Our simulations confirm that the reduced accessibility to conformations geared to an excited state proton transfer may be responsible for the fluorescence in IFP, just as has been suggested by Kennis et al. (Proc. Natl. Acad. Sci. U.S.A., 2010, 107, 9170-9175) for fluorescent bacteriophytochrome from Rhodopseudomonas palustris.