Analyzing protein circular dichroism spectra for accurate secondary structures

Johnson, W. Curtis

doi:10.1002/(sici)1097-0134(19990515)35:3<307::aid-prot4>3.0.co;2-3

Cited by 652 publications

(490 citation statements)

References 28 publications

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“…The protein concentration of 37.7 M was determined via quantitative amino acid analysis. Helical content was estimated with the secondary structure analysis programs Selcon3 (30,31), CDSSTR (30,32), and CONTIN (32,33). (35) to generate five models of the D. discoideum MTBD.…”

Section: Methodsmentioning

confidence: 99%

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

McNaughton

Tikhonenko

Banavali

et al. 2010

Journal of Biological Chemistry

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Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ϳ10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained ␤ ؉ registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691-1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil ␤ ؉ registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the ␣ and ␤ ؉ registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state.Dyneins comprise one of the three families of cytoskeletonbased molecular motors that generate force and translocate cargo in eukaryotic cells (2). These motors work by coupling high and low affinity binding to microtubule or actin filaments, with force producing conformational changes driven by an ATP catalytic cycle (3-5). The coordination between these steps is critical for efficient linear movement. Force production occurs after tight binding is achieved, in a manner that both moves cargo forward and facilitates motor repositioning to advance another step. Although ATP hydrolysis and product release provide the thermodynamic driving force for motility, binding to the microtubule or actin filaments also provides feedback to the catalytic pocket and influences the catalytic rate.An understanding of how substrate affinity is achieved and how it is coupled to nucleotide hydrolysis remains an important problem for all three families of motors (dynein, kinesin, and myosin). For dynein, these issues are particularly complex. In contrast to the close proximity of the substrate-binding domains and catalytic sites in kinesin or myosin-type motors (5), the ATP-sensitive interaction of dynein with microtubules occurs through contacts within a relatively small (ϳ125 ...

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Section: Methodsmentioning

confidence: 99%

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

McNaughton

Tikhonenko

Banavali

et al. 2010

Journal of Biological Chemistry

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“…CD signals were converted to mean residue molar ellipticities and secondary structure content was determined using the DichroWeb 23 software package CDSSTR. 35 The thermal transitions were determined by plotting the second derivative of the thermal unfolding curves and identifying the inflection point of each transition (OriginPro 8.6.0).…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

et al. 2013

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The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein.We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligooxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,Ndimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; CMC, critical micelle concentration; DGS-NTA, 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl]; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidylglycerol; DSP, dithiobis[succinimidyl proprionate; FM, fluorescein maleimide; FRET, F-rster resonance energy transfer; Ipa, invasion plasmid antigen; Ipg, invasion plasmid gene; LDAO, N,N-dimethyldodecylamine N-oxide; Mxi, major exporter of Ipas; OPOE, n-Octyl-oligo-oxyethylene; SATA, N-succinimidyl-S-acetylthioacetate; SEC, size exclusion chromatography; SRB, sulforhodamine B; T m , thermal unfolding midpoint; TCEP, (tris (2-carboxyethyl)phosphine; TRITC DHPE, (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine); T3SS, type-III secretion system; T3SA, type III secretion apparatus.Additional Supporting Information may be found in the online version of this article. that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.

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“…1b). But even after 15 min at pH 1.7, the gC1q protomers did not have a CD-spectrum typical of a fully denaturated protein and retained some residual secondary structure [26,27]. The presence of a disulfide bridge in each chain of gC1q (chain A: C150-C168, chain B: C154-C171, chain C: C151-C165) might be responsible for the residual secondary structure in the dissociated molecule.…”

Section: Disassembly and Unfolding Of The Gc1q Trimermentioning

confidence: 99%

Trimeric reassembly of the globular domain of human C1q

Tacnet

Cheong

Goeltz

et al. 2008

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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C1q is a versatile recognition protein which binds to a variety of targets and consequently triggers the classical pathway of complement. C1q is a hetero-trimer composed of three chains (A, B and C) arranged in three domains, a short N-terminal region, followed by a collagenous repeat domain that gives rise to the formation of (ABC) triple helices, each ending in a C-terminal hetero-trimeric globular domain, called gC1q, which is responsible for the recognition properties of C1q. The mechanism of the trimeric assembly of C1q and in particular the role of each domain in the process is unknown. Here, we have investigated if the gC1q domain was able to assemble into functional trimers, in vitro, in the absence of the collagenous domain, a motif known to promote obligatory trimers in other proteins. Acid-mediated gC1q protomers reassembled into functional trimers, once neutralized, indicating that it is the gC1q domain which possesses the information for trimerization. However, reassembly occurred after neutralization, only if the gC1q protomers had preserved a residual tertiary structure at the end of the acidic treatment. Thus, the collagenous domain of C1q might initialize the folding of the gC1q domain so that subsequent assembly of the entire molecule can occur.

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Analyzing protein circular dichroism spectra for accurate secondary structures

Cited by 652 publications

References 28 publications

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

Trimeric reassembly of the globular domain of human C1q

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