New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins.

Arun K Attri; Allen P Minton

doi:10.1016/j.ab.2004.09.045

Home/Archive/PubMed

PubMed · 15649382

New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins.

Arun K Attri·Allen P Minton

View original source ↗Export metadata ↓

Abstract

A method for rapid detection and characterization of reversible associations of macromolecules in solution is presented. A programmable dual-syringe infusion pump is used to introduce a solution of time-varying composition into parallel flow cells for concurrent measurement of laser light scattering at multiple angles and ultraviolet-visible absorbance. An experiment lasting less than 15 min produces a large and information-rich set of data, consisting of several thousand values of the Rayleigh ratio as a function of solute concentration(s) and scattering angle. Using a novel treatment of the data, the entire data set may be equally rapidly analyzed in the context of models for self-association. Validation experiments conducted on previously characterized nonassociating and self-associating proteins yielded robust values for molecular weights in the range 10-330 kDa and equilibrium association constants for dimer formation in the range 2 x 10(3)-6 x 10(5) M(-1).

Explore related subjects

Dimerization Equipment Design Light Models, Theoretical Multiprotein Complexes Proteins Scattering, Radiation Solutions Journal Article Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Arun K Attri, Allen P Minton. 2005-02-01. New methods for measuring macromolecular interactions in solution via static light scattering: basic methodology and application to nonassociating and self-associating proteins.. https://doi.org/10.1016/j.ab.2004.09.045

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

A designed well-folded monomeric four-helix bundle protein prepared by Fmoc solid-phase peptide synthesis and native chemical ligation.

The design and total chemical synthesis of a monomeric native-like four-helix bundle protein is presented. The designed protein, GTD-Lig, consists of 90 amino acids and is based on the dimeric structure of the de novo designed helix-loop-helix GTD-43. GTD-Lig was prepared by the native chemical ligation strategy and the fragments (45 residues long) were synthesized by applying standard fluorenylmethoxycarbonyl (Fmoc) chemistry. The required peptide-thioester fragment was prepared by anchoring the free gamma-carboxy group of Fmoc-Glu-allyl to the solid phase. After chain elongation the allyl moiety was orthogonally removed and the resulting carboxy group was functionalized with a glycine-thioester followed by standard trifluoroacetic acid (TFA) cleavage to produce the unprotected peptide-thioester. The structure of the synthetic protein was examined by far- and near-UV circular dichroism (CD), sedimentation equilibrium ultracentrifugation, and NMR and fluorescence spectroscopy. The spectroscopic methods show a highly helical and native-like monomeric protein consistent with the design. Heat-induced unfolding was studied by tryptophan absorbance and far-UV CD. The thermal unfolding of GTD-Lig occurs in two steps; a cooperative transition from the native state to an intermediate state and thereafter by noncooperative melting to the unfolded state. The intermediate exhibits the properties of a molten globule such as a retained native secondary structure and a compact hydrophobic core. The thermodynamics of GuHCl-induced unfolding were evaluated by far-UV CD monitoring and the unfolding exhibited a cooperative transition that is well-fitted by a two-state mechanism from the native to the unfolded state. GTD-Lig clearly shows the characteristics of a native protein with a well-defined structure and typical unfolding transitions. The design and synthesis presented herein is of general applicability for the construction of large monomeric proteins.

Dimerization↗

PubMed2006

Recombinant hemoglobin betaG83C-F41Y.

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb betaG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.

Dimerization↗

PubMed2005

Leukemogenic MLL fusion proteins bind across a broad region of the Hox a9 locus, promoting transcription and multiple histone modifications.

Chromosome translocations involving the mixed lineage leukemia gene MLL are associated with aggressive acute leukemias in both children and adults. Leukemogenic MLL fusion proteins delete the MLL SET domain Lys(4) methyltransferase activity and fuse MLL to 1 of >40 different translocation partners. Some MLL fusion proteins involve nuclear proteins that are transcriptional activators, whereas others have transcriptional activating activity but instead dimerize the truncated MLL molecule. Both types of MLL fusion proteins enforce persistent expression of Hox a9 and Meis1, which is pivotal for leukemogenesis through mechanisms that remain obscure. Here, we show that nuclear and dimerizable forms of MLL bind with a similar pattern to the Hox a9 locus that overlaps the distribution of wild-type MLL and deregulate transcription of three isoforms of Hox a9. Induction of MLL fusion protein activity is associated with increased levels of histone acetylation and Lys(4) methylation at Hox target genes. In addition, the MLL-ENL-ER protein, but not dimerized MLL, also induces dimethylation of histone H3 at Lys(79), suggesting alternative mechanisms for transcriptional activation.

Dimerization↗