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ABSTRACT: HIV-1 enzyme reverse transcriptase (RT) is a major target for antiviral drug development, with over half of current FDA-approved therapeutics against HIV infection targeting the DNA polymerase activity of this enzyme. HIV-1 RT is a multifunctional enzyme that has RNA and DNA dependent polymerase activity, along with ribonuclease H (RNase H) activity. The latter is responsible for degradation of the viral genomic RNA template during first strand DNA synthesis to allow completion of reverse transcription and the viral dsDNA. While the RNase H activity of RT has been shown to be essential for virus infectivity, all currently used drugs directed at RT inhibit the polymerase activity of the enzyme; none target RNase H. In the last decade, the increasing prevalence of HIV variants resistant to clinically used antiretrovirals has stimulated the search for inhibitors directed at stages of HIV replication different than those targeted by current drugs. HIV RNase H is one such novel target and, over the past few years, significant progress has been made in identifying and characterizing new RNase H inhibitor pharmacophores. In this review we focus mainly on the most potent low micromolar potency compounds, as these provide logical bases for further development. We also discuss why HIV RNase H has been a difficult target for antiretroviral drug development.
Biology. 10/2012; 1(3):521-541.
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ABSTRACT: In the rapidly evolving disease of HIV drug resistance readily emerges, nullifying the effectiveness of therapy. Drug resistance has been extensively studied in HIV-1 protease where resistance occurs when the balance between enzyme inhibition and substrate recognition and turn-over is perturbed to favor catalytic activity. Mutations which confer drug resistance can impact the dynamics and structure of both the bound and unbound forms of the enzyme. Flap+ is a multi-drug-resistant variant of HIV-1 protease with a combination of mutations at the edge of the active site, within the active site, and in the flaps (L10I, G48V, I54V, V82A). The impact of these mutations on the dynamics in the unliganded form in comparison with the wild-type protease was elucidated with Molecular Dynamic simulations and NMR relaxation experiments. The comparative analyses from both methods concur in showing that the enzyme's dynamics are impacted by the drug resistance mutations in Flap+ protease. These alterations in the enzyme dynamics, particularly within the flaps, likely modulate the balance between substrate turn-over and drug binding, thereby conferring drug resistance.
Journal of Chemical Theory and Computation 10/2012; 8(10):3452-3462. · 5.22 Impact Factor
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ABSTRACT: HIV-1 reverse transcriptase (RT) has been an attractive target for the development of antiretroviral agents. Although this enzyme is bi-functional, having both DNA polymerase and ribonuclease H (RNH) activities, there is no clinically approved inhibitor of the RNH activity. Here, we characterize the structural basis and molecular interaction of an allosteric site inhibitor, BHMP07, with the wild-type (WT) RNH fragment. Solution NMR experiments for inhibitor titration on WT RNH showed relatively wide chemical shift perturbations, suggesting a long-range conformational effect on the inhibitor interaction. Comparisons of the inhibitor-induced NMR chemical shift changes of RNH with those of RNH dimer, in the presence and absence of Mg(2+) , were performed to determine and verify the interaction site. The NMR results, with assistance of molecular docking, indicate that BHMP07 preferentially binds to a site that is located between the RNH active site and the region encompassing helices B and D (the 'substrate-handle region'). The interaction site is consistent with the previous proposed site, identified using a chimeric RNH (p15-EC) [Gong et al. (2011) Chem Biol Drug Des 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH.
Chemical Biology & Drug Design 07/2012; 80(5):706-16. · 2.28 Impact Factor
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ABSTRACT: Nitrogen-15 Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation experiment are widely used to characterize protein backbone dynamics and chemical exchange parameters. Although an accurate value of the transverse relaxation rate, R(2), is needed for accurate characterization of dynamics, the uncertainty in the R(2) value depends on the experimental settings and the details of the data analysis itself. Here, we present an analysis of the impact of CPMG pulse phase alternation on the accuracy of the (15)N CPMG R(2). Our simulations show that R(2) can be obtained accurately for a relatively wide spectral width, either using the conventional phase cycle or using phase alternation when the r.f. pulse power is accurately calibrated. However, when the r.f. pulse is miscalibrated, the conventional CPMG experiment exhibits more significant uncertainties in R(2) caused by the off-resonance effect than does the phase alternation experiment. Our experiments show that this effect becomes manifest under the circumstance that the systematic error exceeds that arising from experimental noise. Furthermore, our results provide the means to estimate practical parameter settings that yield accurate values of (15)N transverse relaxation rates in the both CPMG experiments.
Journal of Biomolecular NMR 04/2012; 53(1):13-23. · 3.61 Impact Factor
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Rieko Ishima
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ABSTRACT: Nuclear Magnetic Resonance (NMR) relaxation is a powerful technique that provides information about internal dynamics associated with configurational energetics in proteins, as well as site-specific information involved in conformational equilibria. In particular, (15)N relaxation is a useful probe to characterize overall and internal backbone dynamics of proteins because the relaxation mainly reflects reorientational motion of the N-H bond vector. Over the past 20 years, experiments and protocols for analysis of (15)N R (1), R 2, and the heteronuclear (15)N-{(1)H} NOE data have been well established. The development of these methods has kept pace with the increase in the available static-magnetic field strength, providing dynamic parameters optimized from data fitting at multiple field strengths. Using these methodological advances, correlation times for global tumbling and order parameters and correlation times for internal motions of many proteins have been determined. More recently, transverse relaxation dispersion experiments have extended the range of NMR relaxation studies to the milli- to microsecond time scale, and have provided quantitative information about functional conformational exchange in proteins. Here, we present an overview of recent advances in (15)N relaxation experiments to characterize protein backbone dynamics.
Topics in current chemistry 09/2011; 326:99-122. · 4.29 Impact Factor
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ABSTRACT: Sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is essential for muscle function by transporting Ca(2+) from the cytosol into the sarcoplasmic reticulum through ATP hydrolysis. In this report, the effects of substitution mutations on the isolated SERCA-nucleotide binding domain (SERCA-N) were studied using NMR. (15)N-(1)H HSQC spectra of substitution mutants at the nucleotide binding site, T441A, R560V, and C561A, showed chemical shift changes, primarily in residues adjacent to the mutation sites, indicating only local effects. Further, the patterns of chemical shift changes upon AMP-PNP binding to these mutants were similar to that of the wild type SERCA-N (WT). In contrast to these nucleotide binding site mutants, a mutant found in patients with Darier's disease, E412G, showed small but significant chemical shift changes throughout the protein and rapid precipitation. However, the AMP-PNP dissociation constant (∼2.5 mM) was similar to that of WT (∼3.8 mM). These results indicate that the E412G mutant retains its catalytic activity but most likely reduces its stability. Our findings provide molecular insight into previous clinical, physiological, and biochemical observations.
Biochemical and Biophysical Research Communications 02/2011; 405(1):19-23. · 2.48 Impact Factor
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ABSTRACT: HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors.
Chemical Biology & Drug Design 01/2011; 77(1):39-47. · 2.28 Impact Factor
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ABSTRACT: Modular proteins contain individual domains that are often connected by flexible, unstructured linkers. Using a model system based on the GB1 domain, we constructed tandem repeat proteins and investigated the rotational diffusion and long-range angular ordering behavior of individual domains by measuring NMR relaxation parameters and residual dipolar couplings. Although they display almost identical protein-solvent interfaces, each domain exhibits distinct rotational diffusion and alignment properties. The diffusion tensor anisotropy of the N-terminal domain (NTD) is D(‖)/D(⊥) = 1.5-1.6, similar to that of single-GB1 domains (D(‖)/D(⊥) = 1.6-1.7), whereas the value for the C-terminal domain (CTD) is D(‖)/D(⊥) = 2.0-2.2. In addition, the two domains have different rotational correlation times. These effects are observed for linkers of three to 24 residues, irrespective of linker length. The NTD and CTD also differ in their degree of magnetic alignment, even with a flexible linker of 18 residues, exhibiting D(a) values of 7.7 Hz and 9.7 Hz, respectively. Our results suggest that diffusion differences and long-range influences may persist in modular protein systems, even for systems that have highly flexible linkers and exhibit no domain-domain or domain-linker interactions.
Biophysical Journal 10/2010; 99(8):2636-46. · 3.65 Impact Factor
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ABSTRACT: Membrane proteins pose problems for the application of NMR-based ligand-screening methods because of the need to maintain the proteins in a membrane mimetic environment such as detergent micelles: they add to the molecular weight of the protein, increase the viscosity of the solution, interact with ligands non-specifically, overlap with protein signals, modulate protein dynamics and conformational exchange and compromise sensitivity by adding highly intense background signals. In this article, we discuss the special considerations arising from these problems when conducting NMR-based ligand-binding studies with membrane proteins. While the use of (13)C and (15)N isotopes is becoming increasingly feasible, (19)F and (1)H NMR-based approaches are currently the most widely explored. By using suitable NMR parameter selection schemes independent of or exploiting the presence of detergent, (1)H-based approaches require least effort in sample preparation because of the high sensitivity and natural abundance of (1)H in both, ligand and protein. On the other hand, the (19)F nucleus provides an ideal NMR probe because of its similarly high sensitivity to that of (1)H and the lack of natural (19)F background in biologic systems. Despite its potential, the use of NMR spectroscopy is highly underdeveloped in the area of drug discovery for membrane proteins.
Chemical Biology & Drug Design 03/2010; 75(3):237-56. · 2.28 Impact Factor
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ABSTRACT: Purification and in vitro protein-folding schemes were developed to produce monodisperse samples of the mature wild-type HIV-2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2(E37K) (elevated K(d) approximately 20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a T(m) of 60.5 for PR2 as compared with 65.7 degrees C for HIV-1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the T(m) of PR2 increases by 14.8 degrees C in the presence of DRV as compared with 22.4 degrees C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2(T26A)) or a deletion of the C-terminal residues 96-99 (PR2(1-95)), drastically increase the K(d) (>10(5)-fold). PR2(T26A) and PR2(1-95) consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size-exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild-type PR2 and its active-site mutant PR2(D25N) are folded dimers. Addition of twofold excess active-site inhibitor promotes dimerization of PR2(T26A) but not of PR2(1-95), indicating that subunit interactions involving the C-terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.
Protein Science 10/2009; 18(12):2442-53. · 2.80 Impact Factor
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ABSTRACT: The structural and functional role of conserved residue G86 in HIV-1 protease (PR) was investigated by NMR and crystallographic analyses of substitution mutations of glycine to alanine and serine (PR(G86A) and PR(G86S)). While PR(G86S) had undetectable catalytic activity, PR(G86A) exhibited approximately 6000-fold lower catalytic activity than PR. (1)H-(15)N NMR correlation spectra revealed that PR(G86A) and PR(G86S) are dimeric, exhibiting dimer dissociation constants (K(d)) of approximately 0.5 and approximately 3.2 muM, respectively, which are significantly lower than that seen for PR with R87K mutation (K(d) > 1 mM). Thus, the G86 mutants, despite being partially dimeric under the assay conditions, are defective in catalyzing substrate hydrolysis. NMR spectra revealed no changes in the chemical shifts even in the presence of excess substrate, indicating very poor binding of the substrate. Both NMR chemical shift data and crystal structures of PR(G86A) and PR(G86S) in the presence of active-site inhibitors indicated high structural similarity to previously described PR/inhibitor complexes, except for specific perturbations within the active site loop and around the mutation site. The crystal structures in the presence of the inhibitor showed that the region around residue 86 was connected to the active site by a conserved network of hydrogen bonds, and the two regions moved further apart in the mutants. Overall, in contrast to the role of R87 in contributing significantly to the dimer stability of PR, G86 is likely to play an important role in maintaining the correct geometry of the active site loop in the PR dimer for substrate binding and hydrolysis. Proteins 2010. (c) 2009 Wiley-Liss, Inc.
Proteins Structure Function and Bioinformatics 09/2009; 78(4):1015-25. · 3.39 Impact Factor
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ABSTRACT: In the analysis of the constant-time Carr-Purcell-Meiboom-Gill (CT-CPMG) relaxation dispersion experiment, chemical exchange parameters, such as rate of exchange and population of the exchanging species, are typically optimized using equations that predict experimental relaxation rates recorded as a function of effective field strength. In this process, the effect of chemical exchange during the CPMG pulses is typically assumed to be the same as during the free-precession. This approximation may introduce systematic errors into the analysis of data because the number of CPMG pulses is incremented during the constant-time relaxation period, and the total pulse duration therefore varies as a function of the effective field strength. In order to estimate the size of such errors, we simulate the time-dependence of magnetization during the entire constant time period, explicitly taking into account the effect of the CPMG pulses on the spin relaxation rate. We show that in general the difference in the relaxation dispersion profile calculated using a practical pulse width from that calculated using an extremely short pulse width is small, but under certain circumstances can exceed 1 s(-1). The difference increases significantly when CPMG pulses are miscalibrated.
Journal of Biomolecular NMR 08/2009; 45(1-2):207-16. · 3.61 Impact Factor
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Rui Gong,
Bang K Vu,
Yang Feng,
DaRue A Prieto,
Marzena A Dyba,
Joseph D Walsh,
Ponraj Prabakaran,
Timothy D Veenstra,
Sergey G Tarasov, Rieko Ishima,
Dimiter S Dimitrov
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ABSTRACT: The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.
Journal of Biological Chemistry 04/2009; 284(21):14203-10. · 4.77 Impact Factor
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ABSTRACT: The 15N transverse relaxation as measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence is often used to characterize protein backbone dynamics in solution. In the 15N CPMG experiment, the delay between 15N CPMG pulses typically is set sufficiently short to minimize the effects of relaxation by the 15N-1H antiphase component. This limits the total relaxation duration of the CPMG experiment because frequent application of the high B1 field to the heteronucleus may cause probe damage. The resulting antiphase component can be minimized by using 1H composite decoupling or by applying 1H 180° pulses more frequently than the 15N CPMG pulses. In this study, we simulated 15N CPMG relaxation in the presence of either 1H composite decoupling or 1H 180° pulses and compared the results with the experimental transverse relaxation data acquired with [u-15N]- or [u-15N, u-2H]-ubiquitin samples. We explain systematic errors resulting from these CPMG experiments and describe a method for recording 15N transverse relaxation in a manner that minimizes probe problems. © 2009 Wiley Periodicals, Inc. Concepts Magn Reson Part A 34A: 63–75, 2009.
Concepts in Magnetic Resonance Part A 03/2009; 34A(2):63 - 75. · 1.67 Impact Factor
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ABSTRACT: Information on local dynamics of antibodies is important to evaluate stability, to rationally design variants, and to clarify conformational disorders at the epitope binding sites. Such information may also be useful for improved understanding of antigen recognition. NMR can be used for characterization of local protein dynamics at the atomic level through relaxation measurements. Due to the complexity of the NMR spectra, an extensive use of this method is limited to small protein molecules, for example, antibody domains and some scFv. Here, we describe a protocol that was used to study the dynamics of an antibody domain in solution using NMR. We describe protein preparation for NMR studies, NMR sample optimization, signal assignments, and dynamics experiments.
Methods in molecular biology (Clifton, N.J.) 02/2009; 525:533-43, xv.
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ABSTRACT: An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR(RE) containing Asp25Arg and Gly49Glu mutations, and PR(RER) containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. (1)H-(15)N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR(RE) has a high tendency to aggregate, PR(RER) exists mainly as a folded monomer at 25-35 microM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR(RE) sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR(RER) sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR(RER) protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.
Protein Engineering Design and Selection 08/2008; 21(7):453-61. · 2.94 Impact Factor
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ABSTRACT: All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR(D25N)) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 +/- 0.09 microm) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR(D25N) in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 degrees C, respectively. Only minimal differences are observed in high resolution crystal structures of PR(D25N) complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH(2) (RPB), as compared with PR.DRV and PR.RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T(m) is smaller for PR(D25N) (6.2 degrees C) than for PR (8.7 degrees C). The T(m) of PR(D25N).DRV increases by only 3 degrees C relative to free PR(D25N), as compared with a 22 degrees C increase for PR.DRV, and the mutation increases the ligand dissociation constant of PR(D25N).DRV by a factor of approximately 10(6) relative to PR.DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.
Journal of Biological Chemistry 06/2008; 283(19):13459-70. · 4.77 Impact Factor
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ABSTRACT: The Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion NMR experiment is a powerful means for detecting and characterizing conformational exchange. This experiment reports the exchange of chemical shifts and therefore can monitor all chemical exchange phenomena, not only intramolecular conformational exchange. Here, we report a CPMG transverse relaxation dispersion study for the monomer-dimer equilibrium of the GB1 point mutant, Ala-34-Phe (GB1(A34F)). This variant exists predominantly as a side-by-side dimer at high concentration (>1 mM). We demonstrate that the dispersion experiment is exceptionally valuable for studying association equilibria since it is extremely sensitive to the minor population in the equilibrium. Twenty-eight individual amide sites in the GB1(A34F) dimer protein were monitored via a 2D (15)N-(1)H HSQC spectroscopy, and all relaxation-derived data are consistent with predominantly an exchange process between dimer and monomer species.
The Journal of Physical Chemistry B 05/2008; 112(19):6008-12. · 3.70 Impact Factor
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ABSTRACT: Internal motion in proteins fulfills a multitude of roles in biological processes. NMR spectroscopy has been applied to elucidate protein dynamics at the atomic level, albeit at a low resolution, and is often complemented by molecular dynamics simulation. However, it is critical to justify the consistency between simulation results and conclusions often drawn from multiple experiments in which uncertainties arising from assumed motional models may not be explicitly evaluated. To understand the role of the flaps of HIV-1 protease dimer in substrate recognition and protease function, many molecular dynamics simulations have been performed. The simulations have resulted in various proposed models of the flap dynamics, some of which are more consistent than others with our working model previously derived from experiments. However, using the working model to discriminate among the simulation results is not straightforward because the working model was derived from a combination of NMR experiments and crystal structure data. In this study, we use the NMR chemical shifts and relaxation data of the protease "monomer" rather than structural data to narrow down the possible conformations of the flaps of the "dimer". For the first time, we show that the tips of the flaps in the unliganded protease dimer interact with each other in solution. Accordingly, we discuss the consistency of the simulations with the model derived from all experimental data.
Proteins Structure Function and Bioinformatics 04/2008; 70(4):1408-15. · 3.39 Impact Factor
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ABSTRACT: Protein backbone dynamics is often characterized using model-free analysis of three sets of (15)N relaxation data: longitudinal relaxation rate (R1), transverse relaxation rate (R2), and (15)N-{H} NOE values. Since the experimental data is limited, a simplified model-free spectral density function is often used that contains one Lorentzian describing overall rotational correlation but not one describing internal motion. The simplified spectral density function may be also used in estimating the overall rotational correlation time, by making the R2/R1 largely insensitive to internal motions, as well as used as one of the choices in the model selection protocol. However, such approximation may not be valid for analysis of relaxation data of large proteins recorded at high magnetic field strengths since the contribution to longitudinal relaxation from the Lorentzian describing the overall rotational diffusion of the molecule is comparably small relative to that describing internal motion. Here, we quantitatively estimate the errors introduced by the use of the simplified spectral density in model-free analysis for large proteins at high magnetic field strength.
Journal of Biomolecular NMR 09/2007; 38(4):315-24. · 3.61 Impact Factor
