Lysine-specific acetylated proteome from the archaeon Thermococcus gammatolerans reveals the presence of acetylated histones.

Thermococcus gammatolerans EJ3 is an extremophile archaeon which was revealed as one of the most radioresistant organisms known on Earth, withstanding up to 30 kGy gamma-ray radiations. While its theoretical proteome is rather small, T. gammatolerans may enhance its toolbox by post-translational modification of its proteins. Here, we explored its extent of Nε-acetylation of lysines. For this, we immunopurified with two acetylated-lysine antibodies the acetylated peptides resulting from a proteolysis of soluble proteins with trypsin. The comparison of acetylated proteomes of two archaea highlights some common acetylation patterns but only 4 out of 26 orthologous proteins found to be acetylated in both species, are acetylated on the same lysine site. We evidenced that histone B is acetylated in T. gammatolerans at least at two different sites (K27 and K36), and a peptide common at the C-terminus of histones A and B is also acetylated. We verified that acetylation of histones is a common trait among Thermococcales after recording data on Thermococcus kodakaraensis histones and identifying three acetylated sites. This discovery reinforces the strong evolutionary link between Archaea and Eukaryotes and should be an incentive for further investigation on the extent and role of acetylation of histones in Archaea. SIGNIFICANCE: Acetylation is an important post-translational modification of proteins that has been extensively described in Eukaryotes, and more recently in Bacteria. Here, we report for the first time ever that histones in Archaea are also modified by acetylation after a systematic survey of acetylated peptides in Thermococcus gammatolerans. Structural models of histones A and B indicates that acetylation of the identified modified residues may play an important role in histone assembly and/or interaction with DNA. The in-depth protein acetylome landscape in T. gammatolerans includes at least 181 unique protein sequences, some of them being modified on numerous residues. Proteins involved in metabolic processes, information storage and processing mechanisms are over-represented categories in this dataset, highlighting the ancient role of this protein post-translational modification in primitive cells.

Prioritizing targets for structural biology through the lens of proteomics: the archaeal protein TGAM_1934 from Thermococcus gammatolerans.

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron-sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 (http://proteomecentral.proteomexchange.org/dataset/PXD000402) and Protein Data Bank under the accession number 2mcf, respectively.

N- and O-acetylation of threonine residues in the context of proteomics.

The detection of post-translational modifications (PTMs) of proteins is a matter of intensive research. Among all possible pitfalls that may lead to misidentifications, the chemical stability of modified peptides is scarcely questioned. Global proteomic studies devoted to protein acetylation are becoming popular. Thus, we were concerned about the intrinsic stability of O-acetylated peptides because of the O-N acyl transfer reactivity occurring when an amino moiety is present in the vicinity of the acylated hydroxyl group. Here, the behavior of isomeric O- and N-acetylated, N-terminal threonine-containing peptides was explored in a standard proteomic workflow. We demonstrated a strong chemical instability of O-acetylation, which prevents its detection.

Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation.

BACKGROUND: Deinococcus deserti VCD115 has been isolated from Sahara surface sand. This radiotolerant bacterium represents an experimental model of choice to understand adaptation to harsh conditions encountered in hot arid deserts. We analysed the soluble proteome dynamics in this environmentally relevant model after exposure to 3 kGy gamma radiation, a non-lethal dose that generates massive DNA damages. For this, cells were harvested at different time lapses after irradiation and their soluble proteome contents have been analysed by 2-DE and mass spectrometry. RESULTS: In the first stage of the time course we observed accumulation of DNA damage response protein DdrB (that shows the highest fold change ~11), SSB, and two different RecA proteins (RecAP and RecAC). Induction of DNA repair protein PprA, DNA damage response protein DdrD and the two gyrase subunits (GyrA and GyrB) was also detected. A response regulator of the SarP family, a type II site-specific deoxyribonuclease and a putative N-acetyltransferase are three new proteins found to be induced. In a more delayed stage, we observed accumulation of several proteins related to central metabolism and protein turn-over, as well as helicase UvrD and novel forms of both gyrase subunits differing in terms of isoelectric point and molecular weight. CONCLUSIONS: Post-translational modifications of GyrA (N-terminal methionine removal and acetylation) have been evidenced and their significance discussed. We found that the Deide_02842 restriction enzyme, which is specifically found in D. deserti, is a new potential member of the radiation/desiccation response regulon, highlighting the specificities of D. deserti compared to the D. radiodurans model.

Revisiting iodination sites in thyroglobulin with an organ-oriented shotgun strategy.

Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies.

Proteomics-based refinement of Deinococcus deserti genome annotation reveals an unwonted use of non-canonical translation initiation codons.

Deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of the Deinococcus deserti genome, we performed an N terminus-oriented characterization of its proteome. For this, we used a labeling reagent, N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein N termini. The large scale identification of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide-modified N-terminal-most peptides by shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and the correction of 73 translation initiation codons in the D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. We also analyzed signal peptide cleavages on a genome-wide scale. Based on comparative proteogenomics analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We also report the use of non-conventional translation initiation codons for two other genes: Deide_03051 and infC. Whether such use of non-canonical translation initiation codons is much more frequent than for other previously reported bacterial phyla or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.

Palladium motion in cyclomeric compounds: a theoretical study.

Density functional theory (DFT)/B3LYP calculations have been carried out to study successive intramolecular 1,n palladium shifts (n = 3-5) in palladium complexes of organic cyclomers. Such shifts of the PdBr(phosphine) moiety, which is bound to the cyclomer and which exchanges concomitantly with a hydrogen atom distant by n carbon atoms from palladium, might lead to an endless motion around the cyclomer. The cyclomers that have been analyzed are either the [1,1,1,1]paracyclophane 1, a THF-based 16-crown-4 structure 2, or the THF-ethylene cyclic dimer 3 (THF = tetrahydrofuran). We show that the [1,1,1,1]paracyclophane 1 is not a good candidate for a circular motion of the metallic moiety. This is due to the very high barrier of the 1,2 Pd/H exchange within the phenyl ring. Hence only a pendulum movement of the metallic moiety between two adjacent phenyl rings can easily take place. For the THF-based systems 2 and 3, the processes along the exo face are found to involve high energy barriers. Processes along the endo face are more accessible, especially for 2 where an endless motion made of successive 1,2 and 1,5 shifts is characterized by barriers that are somewhat less than 30 kcal mol(-1).

Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry.

To improve our knowledge on protein targets of uranyl ion (UO(2)(2+)), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO(2)(2+)-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO(2)(2+) ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO(2)(2+)-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.

Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti.

To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.

Autophosphorylated residues involved in the regulation of human chk2 in vitro.

Human checkpoint kinase 2 is a major actor in checkpoint activation through phosphorylation by ataxia telangiectasia mutated in response to DNA double-strand breaks. In the absence of de novo DNA damage, its autoactivation, reported in the event of increased Cds1/checkpoint kinase 2 (Chk2) expression, has been attributed to oligomerization. Here we report a study performed on autoactivated recombinant Chk2 proteins that aims to correlate kinase activity and phosphorylation status. Using a fluorescence-based technique to assay human checkpoint kinase 2 catalytic activity, slight differences in the ability to phosphorylate Cdc25C were observed, depending on the recombinant system used. Using mass spectrometry, the phosphorylation sites were mapped to identify sites potentially involved in the kinase activity. Five phosphorylated positions, at Ser120, Ser260, Thr225, Ser379 and Ser435, were found to be common to bacteria and insect cells expression systems. They were present in addition to the six known phosphorylation sites induced by ionizing radiation (Thr68, Thr432, Thr387, Ser516, Ser33/35 and Ser19) detected by immunoblotting. After phosphatase treatment, Chk2 regained activity via autorephosphorylation. The determination of the five common sites and ionizing-radiation-inducible positions as rephosphorylated confirms that they are potential positive regulators of Chk2 kinase activity. For Escherichia coli's most highly phosphorylated 6His-Chk2, 13 additional phosphorylation sites were assigned, including 7 novel sites on top of recently reported phosphorylation sites.

Specific capture of uranyl protein targets by metal affinity chromatography.

To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.

Through-space intramolecular palladium rearrangement in substituted aryl complexes: theoretical study of the aryl to alkylpalladium migration process.

DFT/B3LYP calculations have been carried out to study intramolecular 1,n palladium shifts (n = 3-5) between sp2 and sp3 carbon atoms in alkylarylpalladium systems. Such shifts, which also involve a concomitant exchange with a hydrogen atom of the alkylaryl ligand, are quite often a pivotal step of several organic transformations mediated by palladium complexes. We show that the intimate mechanism for the 1,3 shift corresponds to a Pd(IV) pathway, whereas a Pd(II) pathway is favored in the case of 1,5 migrations. In the case of 1,4 migrations, both mechanisms are competitive. The Pd(IV) pathway can involve either a true Pd(IV) intermediate (oxidative addition/reductive elimination mechanism) or a Pd(IV) transition state (oxidative hydrogen migration mechanism). The energy barrier is very high for the 1,3 palladium shift, making this process very unlikely, in contrast to the other ones which have enthalpy barriers ranging between 22.8 kcal mol-1 (for the 1,5 shift) and 31.9 kcal mol-1 (for the least favorable 1,4 shift studied here). All of these results are in line with our previous results for palladium shifts between two sp2 carbon atoms. In addition, the sp2 to sp3 shifts have been found to be rather exothermic owing to the possibility for the alkylaryl ligand in the product to achieve a eta3 coordination mode. This eta3 coordination mode results either from the shift itself (1,3 case) or from a subsequent rearrangement that comprises a chain-running mechanism within the alkyl chain bound to the metal (for n > 3).

THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain.

The tRNA:m2(2)G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)--containing N-terminal domain [1-152] and C-terminal catalytic domain [157-329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPalpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPalpha and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPalpha structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNA(Asp) substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.

Can weak interactions modify the binding properties of a strong nitrogen donor? Unusual N-coordination of a phosphoranylidene-substituted pyrazolone unit towards palladium(II) centres: an experimental and theoretical study.

Selective N(2)-binding of 3-methyl-1-phenyl-4-(triphenylphosphoranylidene)-2-pyrazolin-5-one (L) has been found in two palladium(II) complexes, [PdCl(2)L(2)](2) and [Pd(o-C(6)H(4)CH(2)NMe(2))Cl](3). X-Ray diffraction studies show that the pyrazole rings lie almost perpendicular to the coordination plane. In both complexes the metal atom is located out of the plane defined by the pyrazole ring(s)(dihedral angle between the plane and the Pd-N vector approximately 30 degrees). To investigate the origin of this distortion, a theoretical study was carried out on a simplified model of complex , where a single pyrazolone ligand was replaced by NH(3). From this study it could be inferred that the out-of-plane distortion mainly involves weak, electrostatic interactions between a chlorine atom and an ortho-aromatic H atom of the N(1)-linked phenyl group, as well as between the other chlorine atom and an ortho-aromatic H atom of the PPh(3) group.

Industrial process proteomics: alfalfa protein patterns during wet fractionation processing.

A proteome reference map of major soluble proteins from Medicago sativa (alfalfa) leaves and stems has been established for the first time. Among 195 spots analyzed by mass spectrometry and N-terminal Edman sequencing, 117 spots were unambiguously identified, representing 87 different proteins. Of these 87 proteins, 13 proteins were directly identified from the partial genome of Medicago sativa, 30 from expressed sequenced tags (ESTs) of the model legume Medicago truncatula and 44 from closely relative species by a cross-species protein identification method. The proteome map of Medicago sativa was then set as a reference to study the major high protein content products that are generated during the wet fractionation process of alfalfa green biomass. Using two-dimensional electrophoresis, we studied the variation of the protein patterns at different steps of the industrial-scale process. We clearly show that the process induces significant changes including chemical modifications, proteolytic events, and heat-shock protein responses. Strikingly, a certain level of cellular regulation is conserved during biomass processing, as exemplified by the induction of some heat shock proteins. Finally, all the results obtained in this proteomic study may help to identify novel products and to improve process designs in alfalfa biomass plants.

Screening of human serum proteins for uranium binding.

About 20% of uranyl ions in serum are associated with the protein pool. A few of them such as transferrin have been characterized, but most still have to be identified to obtain a better explanation of the biochemical toxicology and kinetics of uranium. We designed an in vitro sensitive procedure involving a combination of bidimensional chromatography with time-resolved fluorescence, coupled with proteomic analysis, to identify uranium-binding proteins in human serum fractions. Ten novel targets were identified and validated using purified proteins and inductively coupled plasma mass spectrometry. Of these, ceruloplasmin, hemopexin, and two complement proteins displayed the capacity to bind uranium with stoichiometry greater than 1 mole of uranium per mole of protein. Not all of these targets are metalloproteins, suggesting that uranyl ions can use a wide variety of binding sites and coordination strategies. These data provide additional insights into a better understanding of uranium chemical toxicity.

Cyclocarbopalladation involving an unusual 1,5-palladium vinyl to aryl shift as termination step: theoretical study of the mechanism.

A DFT/B3LYP model study has been carried out on the cyclocarbopalladation and on an unusual 1,5 vinyl to aryl palladium shift which are the two first steps of a cyclocarbopalladation-Stille coupling tandem reaction of various gamma-bromopropargylic-1,2 diols with alkenyls or alkynyl stannanes catalyzed by Pd(PPh(3))(4). From the calculations, the active intermediates in the catalytic process appear to bear a single phosphine ligand, the palladium(II) center keeping in all cases a square-planar coordination pattern either through intramolecular binding of the triple bond or via an intramolecular Pd...C(phenyl) interaction. The computation of the various transition states and intermediates for the 1,5 vinyl to aryl palladium shift reveals that the intimate mechanism of this pathway corresponds to a one-step hydrogen transfer between the two negatively charged carbon atoms of the vinyl and phenyl groups. A two-step pathway involving a Pd(IV) intermediate is not likely to occur. This conclusion may apply to other 1,n-palladium shifts which have been experimentally observed in various organometallic transformations.

Deciphering structure and topology of conserved COG2042 orphan proteins.

BACKGROUND: The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization. RESULTS: The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure. CONCLUSION: Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs.

sigma-Bond Metathesis Reactions Involving Palladium(II) Hydride and Methyl Complexes: A Theoretical Assessment.

sigma-Bond metathesis reactions of water and methanol with palladium hydride and methyl complexes to yield hydrogen and methane, respectively, have been studied using ab-initio molecular orbital methods at the second-order Møller-Plesset (MP2) perturbation level. The calculations show that such reactions are feasible and that they can in some instances be competitive with an oxidative addition/reductive elimination sequence. A key factor is the presence of an additional lone pair not engaged in the initial bonding of the metal atom with the reacting water or alcohol. Another important controlling factor is the respective charges of the reacting entities. The importance of the choice and of the disposition of the ancillary ligands in the coordination sphere is emphasized.

Synthetic, Structural, Spectroscopic, and Theoretical Studies of Structural Isomers of the Cluster Pt(3)(&mgr;-PPh(2))(3)Ph(PPh(3))(2). A Unique Example of Core Isomerism in Phosphine Phosphido-Rich Clusters.

Two isomers of the phosphido-bridged platinum cluster Pt(3)(&mgr;-PPh(2))(3)Ph(PPh(3))(2) (2 and 3) have been isolated, and their structures have been solved by single-crystal X-ray diffraction. Compound 2 crystallizes in the orthorhombic space group Cmc2(1) with a = 22.192(10) Å, b = 17.650(9) Å, c = 18.182(8) Å, and Z = 4. Compound 3 crystallizes with 2 molecules of dichloromethane in the monoclinic space group C2/c with a = 21.390(10) Å, b = 18.471(9) Å, c = 19.021(11) Å, beta = 105.27(5) degrees, and Z = 4. The two isomers differ essentially in their metal-metal distances and Pt-(&mgr;-PPh(2))-Pt angles. Thus 2, having an imposed C(s) symmetry, contains a bent chain of metal atoms with two short Pt-Pt distances of 2.758(3) Å and a long separation of 3.586(2) Å. In 3, which has an imposed C(2) symmetry, the metal atoms form an isosceles triangle with two Pt-Pt distances of 2.956(3) Å and one of 3.074(4) Å. These isomers can be smoothly interconverted by changing the crystallization solvents. Solution and solid-state (31)P NMR studies have been performed in order to assign the resonances of the different P nuclei and relate their chemical shifts with their structural environments. Raman spectroscopy was used to assign the nu(Pt-Pt) modes of the two structural isomers. Theoretical studies based on extended Hückel calculations and using the fragment molecular orbital approach show that the isomer with the three medium Pt-Pt distances is slightly more stable, in agreement with earlier theoretical predictions. Cluster core isomerism remains a rare phenomenon, and the present example emphasizes the role and the importance of flexible phosphido bridges in stabilizing clusters as well as the unprecedented features which can be observed in phosphine phosphido-rich metal clusters.