Effective rotational correlation times of proteins from NMR relaxation interference

Lee, Donghan; Hilty, Christian; Wider, Gerhard; Wüthrich, Kurt

doi:10.1016/j.jmr.2005.08.014

Cited by 261 publications

(342 citation statements)

References 26 publications

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“…In folded proteins the effective correlation time relates well to the molecular weight of the protein up to 25 to 30 kDa. 25,26 The evaluation of correlation times allows for relatively accurate estimation of protein molecular weights. 24,27 Therefore, this method can readily be used to monitor protein dimerization and protein interactions in general, based on changes that alter molecular weight.…”

Section: Cxcl8 Dimers Do Not Dissociate Upon Hcxcr1pep Engagementmentioning

confidence: 99%

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

et al. 2014

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Interleukin-8 (CXCL8, IL-8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G-protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N-terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild-type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors.

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Section: Cxcl8 Dimers Do Not Dissociate Upon Hcxcr1pep Engagementmentioning

confidence: 99%

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

et al. 2014

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“…The residue specific rotational correlation times were calculated following the approach of Lee et al [9] except that here we used the average geometry-dependent CSA value and bond-length determined from the solution state [23,24]. If a rigid-body model is assumed, the CCRs ( Figure 2F) agree with a monomeric state of the protein.…”

Section: Evaluation Using Experimental Datamentioning

confidence: 99%

“…2) Sampling-In the simulation, we use uniform sampling with 8 points in total, going from 0 to T. (9) T is the relaxational acquisition time. In case of SCT-CCR, it is also equal to the constant delay.…”

Section: Evaluation By Simulationmentioning

confidence: 99%

“…In certain situations they can be used to retrieve significant structural information [7,8]. Another very common use in protein NMR is the extraction of motional correlation times from the interference of 15 N-1 H DD and 15 N CSA interactions [9,10,11]. Many other spin relaxation measurements are, of course, capable of yielding information on motional correlation times.…”

Section: Introductionmentioning

confidence: 99%

“…The downfield component H α N ± (0.5N ± − H z N ± ) relaxes at the difference of an ACR (λ) and a CCR (η) while the upfield component H β N ± (0.5N ± + H z N ± ) relaxes at the sum. Thus CCR can be obtained by taking the difference of the two rates that are separately measured from build-up curves such as in the spin-state selection (S3E-CCR) [13] and TRACT experiments [9], or from the intensity ratio of the up-and downfield peaks of the 15 N doublets such as in the constant-time IP and IPAP experiments [14]. With the exception of the constanttime IP experiment which is applicable to small systems with minimal signal overlap, generally two independent measurements are required, which consumes spectrometer time and makes the measurement more susceptible to spectrometer instabilities and non-idealities in the pulse sequences.…”

Section: Introductionmentioning

confidence: 99%

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Direct measurement of dipole–dipole/CSA cross-correlated relaxation by a constant-time experiment

Liu

Prestegard

2008

Journal of Magnetic Resonance

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Relaxation rates in NMR are usually measured by intensity modulation as a function of a relaxation delay during which the relaxation mechanism of interest is effective. Other mechanisms are often suppressed during the relaxation delay by pulse sequences which eliminate their effects, or cancel their effects when two data sets with appropriate combinations of relaxation rate effects are added. Cross-correlated relaxation (CCR) involving dipole-dipole and CSA interactions differ from autocorrelated relaxation (ACR) in that the signs of contributions can be changed by inverting the state of one spin involved in the dipole-dipole interaction. This property has been exploited previously using CPMG sequences to refocus CCR while ACR evolves. Here we report a new pulse scheme that instead eliminates intensity modulation by ACR and thus allows direct measurement of CCR. The sequence uses a constant time relaxation period for which the contribution of ACR does not change. An inversion pulse is applied at various points in the sequence to effect a decay that depends on CCR only. A 2-D experiment is also described in which chemical shift evolution in the indirect dimension can share the same constant period. This improves sensitivity by avoiding the addition of a separate indirect dimension acquisition time. We illustrate the measurement of residue specific CCR rates on the non-myristoylated yeast ARF1 protein and compare the results to those obtained following the conventional method of measuring the decay rates of the slow and fast-relaxing 15 N doublets. The performances of the two methods are also quantitatively evaluated by simulation. The analysis shows that the shared constant-time CCR (SCT-CCR) method significantly improves sensitivity.

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Resonance Assignments and Structure Determination of Large and Challenging Proteins

Goodrich

Nichols

Frueh

2014

eMagRes

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NMR has become a method of choice for determining structures of proteins in near-physiological conditions, in particular for dynamic systems. However, in large proteins, line-broadening and spectral overlap often preclude resonance assignment of the many correlation maps needed for structural studies. Various well-described spectroscopic techniques and specific isotopic labeling strategies offer relief from these problems, but it is not always clear which samples should be employed to overcome a particular challenge, nor with which pulse sequences they should be used. Here, we review the challenges associated with assigning large proteins, discuss the many labeling schemes employed to overcome hurdles, and describe associated pulse sequences with focus on both practical concerns and the information content each sequence supplies.

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Effective rotational correlation times of proteins from NMR relaxation interference

Cited by 261 publications

References 26 publications

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

Direct measurement of dipole–dipole/CSA cross-correlated relaxation by a constant-time experiment

Resonance Assignments and Structure Determination of Large and Challenging Proteins

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