The 68 Ge/ 68 Ga generator provides an excellent source of positron-emitting 68 Ga. However, newly available ''ionic'' 68 Ge/ 68 Ga radionuclide generators are not necessarily optimized for the synthesis of 68 Ga-labeled radiopharmaceuticals. The eluates have rather large volumes, a high concentration of H 1 (pH of 1), a breakthrough of 68 Ge, increasing with time or frequency of use, and impurities such as stable Zn(II) generated by the decay of 68 Ga, Ti(IV) as a constituent of the column material, and Fe(III) as a general impurity. Methods: We have developed an efficient route for the processing of generator-derived 68 Ga eluates, including the labeling and purification of biomolecules. Preconcentration and purification of the initial generator eluate are performed using a miniaturized column with organic cationexchanger resin and hydrochloric acid/acetone eluent. The purified fraction was used for the labeling of nanomolar amounts of octreotide derivatives either in pure aqueous solution or in buffers. Results: Using the generator post-eluate processing system, .97% of the initially eluated 68 Ga activity was obtained within 4 min as a 0.4-mL volume of a hydrochloric acid/acetone fraction. The initial amount of 68 Ge(IV) was decreased by a factor of 10 4 , whereas initial amounts of Zn(II), Ti(IV), and Fe(III) were reduced by factors of 10 5 , 10 2 , and 10, respectively. The processed 68 Ga fraction was directly transferred to solutions containing labeling precursors-for example, DOTA-DPhe 1 -Tyr 3 -octreotide (DOTATOC) (DOTA 5 1,4,7,N9, N99,. Labeling yields of .95% were achieved within 10 min. Overall yields reached 70% at 20 min after generator elution relative to the eluted 68 Ga activity, not corrected for decay. Specific activities of 68 Ga-DOTATOC were 50 MBq/nmol using a standard protocol, reaching 450 MBq/nmol under optimized conditions. Conclusion: Processing on a cation-exchanger in hydrochloric acid/acetone media represents an efficient strategy for the concentration and purification of generator-derived 68 Ga(III) eluates. The developed scheme guarantees high yields and safe preparation of injectable 68 Ga-labeled radiopharmaceuticals for routine application and is easy to automate. Thus, it is being successfully used in clinical environments and might contribute to a new direction for clinical PET, which could benefit significantly from the easy and safe availability of the radionuclide generatorderived metallic positron-emitter 68 Ga. Zn. 68 Ga is an excellent positron emitter, with 89% positron branching accompanied by low photon emission (1,077 keV, 3.22%) (1,2). 68 Ge/ 68 Ga radionuclide generators have been the object of development and investigation for almost 50 y. For a recent review on this and other PET radionuclide generator systems see Rösch et al. (3).Today, the most common commercially available 68 Ge/ 68 Ga radionuclide generator is based on a TiO 2 solid phase (Cyclotron Co. Ltd.) (4). The generators are produced with 68 Ge activities of up to 3.7 GBq. ''Ionic'' 68 Ga 31...
The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N-and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 k B T (2.7 kJ/mol) through binding of the CL to the Ndomain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance. T he molecular chaperone Hsp90 (heat shock protein 90) is essential for the folding, maturation, and activation of approximately 10% of the yeast proteome. The set of substrate proteins is structurally and functionally diverse and ranges from telomerase to kinases and transcription factors (1-3). Processing of these substrates requires ATP turnover and large conformational rearrangements within Hsp90 (4-6). Interestingly, these conformational states of yeast Hsp90 are not strictly coupled to the binding of nucleotides (7) but are recognized and regulated by the interaction with cochaperones (8) and substrate proteins (9).Hsp90 is a dimer with each monomer consisting of three domains (N, M, and C). The N-terminal domain comprises the nucleotide binding site, whereas the M-domain is important for the binding of many substrates. The C-terminal domain is mainly responsible for the dimerization of the protein. A long charged linker (CL) region, amino acids 211-272 in yeast, connects the N-and M-domain in eukaryotes (Fig. 1A). The crystal structure of yeast Hsp90 was obtained by partly deleting the CL region and shows a closed, compact conformation in the presence of AMP-PNP [Adenosine 5′-(β,γ-imido)triphosphate] and Sba1/p23 (10). The fact that the CL region is difficult to map structurally led to the assumption that this region is disordered and flexible, thereby enabling the conformational rearrangements of Hsp90 (11,12). Besides the structural indetermination of the CL, its ultimate function remains elusive as well (13). Early studies show that parts of the CL region (amino acids 211-259) are dispensable in yeast (14), whereas more extended deletions affect cell viability, substrate maturation, and regulation by cochaperones (11). These deficiencies can be partially rescued by a short linker consisting of an artificial sequence (11), but its specific amino acid sequence is associated with a gain of function, probably an additional Hsp90 regulatory mechanism (15).Single-molecule experiments have recently provided detailed insight int...
During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.
Folding of small proteins often occurs in a two-state manner and is well understood both experimentally and theoretically. However, many proteins are much larger and often populate misfolded states, complicating their folding process significantly. Here we study the complete folding and assembly process of the 1,418 amino acid, dimeric chaperone Hsp90 using single-molecule optical tweezers. Although the isolated C-terminal domain shows two-state folding, we find that the isolated N-terminal as well as the middle domain populate ensembles of fast-forming, misfolded states. These intradomain misfolds slow down folding by an order of magnitude. Modeling folding as a competition between productive and misfolding pathways allows us to fully describe the folding kinetics. Beyond intradomain misfolding, folding of the full-length protein is further slowed by the formation of interdomain misfolds, suggesting that with growing chain lengths, such misfolds will dominate folding kinetics. Interestingly, we find that small stretching forces applied to the chain can accelerate folding by preventing the formation of crossdomain misfolding intermediates by leading the protein along productive pathways to the native state. The same effect is achieved by cotranslational folding at the ribosome in vivo.misfolding | off-pathway | rough energy landscape | optical tweezers L arge protein machines consist of long amino acid chains, often exceeding many hundreds or even over a thousand residues in length. Although the in vitro folding of small and mediumsized proteins is relatively well understood (1-5), very limited information exists about the complete folding process of such large proteins (6). In general, larger proteins often exhibit a multitude of intermediate and aggregation-prone misfolded states (4, 7). Recently, it has been shown that in multidomain proteins with homologous domains, cross-repeat intermediates can greatly slow down productive folding (8) but little is known about how size effects influence the folding of very large (>500 residues) nonhomologous multidomain proteins.Methods providing dynamic as well as structural information are rare, and many bulk methods often do not provide enough resolution to deal with the multitude of states expected for complex systems such as the aforementioned large protein complexes. Single-molecule force spectroscopy offers kinetic, energetic as well as coarse primary structural information combined with the possibility of actively manipulating systems, making it ideally suited for studying the folding of large proteins (5,(9)(10)(11)(12).In this paper, we study the folding and assembly of the large chaperone machinery heat shock protein 90 from yeast (Hsp90), a protein that needs to fold and self-assemble before it can function as a chaperone in the cell. Hsp90 consists of three domains, the N-terminal domain (N domain, 211 residues), the middle domain (M domain, 266 residues), and the C-terminal domain (C domain, 172 residues). In eukaryotic Hsp90, the N and M domains are connecte...
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