Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy

Yang, Yong

doi:10.1016/s1046-2023(02)00308-0

Cited by 98 publications

(85 citation statements)

References 76 publications

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“…This observation raises the question of whether the bent and unbent populations correlate with the different oligomeric states of MutS proteins. To answer this question, we analyzed the oligomeric states of MutS-DNA complexes using a volume analysis method described previously (22)(23)(24). This analysis reveals that under the conditions of our experiments, both E. coli and Taq MutS exist primarily as dimers and tetramers, with Ϸ50% dimers for E. coli MutS and Ϸ75% dimers for Taq MutS, independent of whether they are bound to DNA (data not shown).…”

Section: Conformations Of Muts-dna Complexes Are Independent Of Oligo-mentioning

confidence: 92%

“…For measuring the intrinsic bending of DNA, circles of 10-nm radii, comparable to the size of MutS proteins in the AFM images, were put at the desired locations. Volume analysis was performed as described (22)(23)(24) The positions of the MutS-binding sites on the DNA templates were determined by measuring the length of the DNA from the intersection of the two extrapolated DNA arms to each end. The binding position was then defined as the ratio of the length of the shorter DNA tract divided by the total contour length.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

DNA bending and unbending by MutS govern mismatch recognition and specificity

Wang

Yang²,

Schofield

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

171

233

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DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion͞deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity. D NA mismatch repair (MMR) is a highly conserved repair pathway targeting mismatched bases that arise through DNA replication errors and during homologous recombination (1-3). Inactivation of MMR genes results in a significant increase in the spontaneous mutation rate and, in humans, a predisposition to cancer (4). Escherichia coli provides the best-understood MMR system and serves as a prototype for the more complicated but homologous eukaryotic systems (5). In E. coli, the proteins MutS, MutL, and MutH are responsible for the initiation of MMR (6). MutS and MutL function as dimers and have intrinsic ATPase activities that are essential for MMR (7,8). MMR is initiated by the binding of MutS to either a mismatch or a short insertion͞deletion loop (IDL). Subsequently, ATP induces a conformational change in the MutS-mismatch complex and promotes its interaction with MutL. Assembly of the MutS-MutL-heteroduplex complex activates the endonuclease activity of MutH, which incises the newly synthesized (unmethylated) strand at a d(GATC) site. This incision confers strand specificity of MMR, directing repair exclusively to the newly synthesized strand containing the error. Excision repair completes the process.Crystal structures of E. coli and Thermus aquaticus (Taq) MutS dimers complexed with a G͞T base-base mismatch and a 1T-bulge, respectively, shed light on the structural components of mismatch recognition (9-11). Specific interactions include an aromatic ring stack of a conserved phenylalanine (Phe-39 in Taq or Phe-36 in...

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Section: Conformations Of Muts-dna Complexes Are Independent Of Oligo-mentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

DNA bending and unbending by MutS govern mismatch recognition and specificity

Wang

Yang²,

Schofield

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

171

233

View full text Add to dashboard Cite

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“…Volume analysis of protein peaks was conducted with Image SXM 194-2X (Steve Barrett, University of Liverpool, UK) as described (30). Protein molecular mass was converted into predicted AFM volume using the following equation: V ϭ 1.2⅐M Ϫ 14.7, where V is AFM volume in nm 3 , and M is molecular mass in kDa (31).…”

Section: Methodsmentioning

confidence: 99%

“…Volume analysis of AFM images has been shown to be a powerful tool for determining the oligomerization states of proteins, because the AFM volume depends linearly on the molecular weight of the protein (30,31). ANGPTL4, LPL, and LPL-ANGPTL4 were imaged.…”

Section: Angptl4 Is Not a Catalytic Inhibitor Of Lpl-mentioning

confidence: 99%

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase

Lafferty

Bradford

Erie

et al. 2013

Journal of Biological Chemistry

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Background: Lipoprotein lipase (LPL) clears triglycerides from the blood, and angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. Results: Inhibited LPL is in a complex with ANGPTL4, and upon dissociation LPL regains activity. Conclusion: ANGPTL4 is a reversible, noncompetitive inhibitor of LPL, not an unfolding molecular chaperone as reported previously. Significance: Understanding the mechanism of LPL inhibition supports efforts to develop new therapies for hypertriglyceridemia.

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“…The heights of all identified particles were determined with Digital Instruments off-line section analysis routines. Previous studies have identified the relative height distortion caused by mica substrate surface interactions (27), and molecular mass was determined by comparing calculated particle size to known dendrimer and protein complex standards (27,28).…”

Section: Reconstitution Of Gr Heterocomplexes Containing Tubulin-mousementioning

confidence: 99%

Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein

Harrell

Murphy

Morishima

et al. 2004

Journal of Biological Chemistry

112

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Rapid, ligand-dependent movement of glucocorticoid receptors (GR) from cytoplasm to the nucleus is hsp90-dependent, and much of the movement system has been defined. GR⅐hsp90 heterocomplexes isolated from cells contain one of several hsp90-binding immunophilins that link the complex to cytoplasmic dynein, a molecular motor that processes along microtubular tracks to the nucleus. Although it is known that rapid, hsp90-dependent GR movement requires intact microtubules, it has not been shown that the movement is dynein-dependent. Here, we show that overexpression of dynamitin, which blocks movement by dissociating the dynein motor from its cargo, inhibits ligand-dependent movement of the GR to the nucleus. We show that native GR⅐hsp90⅐immnunophilin complexes contain dynamitin as well as dynein and that GR heterocomplexes isolated from cytosol containing paclitaxel and GTP to stabilize microtubules also contain tubulin. The complete movement system, including the dynein motor complex and tubulin, can be assembled under cell-free conditions by incubating GR immune pellets with paclitaxel/GTP-stabilized cytosol prepared from GR ؊ L cells. This is the first evidence that the movement of a steroid receptor is dynein-dependent, and it is the first isolation of a steroid receptor bound to the entire system that determines its retrograde movement.As the initial step in their action, transcription factors, such as steroid receptors, p53, and HSF1, must move in a targeted manner through the cytoplasm to the nucleus. Until recently, there has been little mechanistic understanding of how protein solutes (i.e. non-vesicle-associated proteins) undergo such retrograde trafficking. Because the glucocorticoid receptor (GR)

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Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy

Cited by 98 publications

References 76 publications

DNA bending and unbending by MutS govern mismatch recognition and specificity

DNA bending and unbending by MutS govern mismatch recognition and specificity

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase

Evidence for Glucocorticoid Receptor Transport on Microtubules by Dynein

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