Rapid measurement of 3J(HN–Hα) and 3J(N–Hβ) coupling constants in polypeptides

Barnwal, Ravi Pratap; Rout, Ashok K.; Chary, Kandala V. R.; Atreya, Hanudatta S.

doi:10.1007/s10858-007-9200-8

Cited by 29 publications

(22 citation statements)

References 23 publications

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“…On the other hand, GFT NMR is a powerful technique for speeding up NMR data acquisition and has been shown to be useful in various applications [2,18,19,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. In the present study, the application of two fast NMR methods, namely GFT NMR and non-uniform sampling to selectively figure (a,b) correspond to Ω( 15 N i+1 + κ * 13 C i+1 ) and Ω( 15 N i+1 − κ * 13 C i+1 ), respectively.…”

Section: Discussionmentioning

confidence: 79%

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

2017

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Abstract:In recent years, there has been a growing interest in fast acquisition and analysis of nuclear magnetic resonance (NMR) spectroscopy data for high throughput protein structure determination. Towards this end, rapid data collection techniques and methods to simplify the NMR spectrum such as amino acid selective unlabeling have been proposed recently. Combining these two approaches can speed up further the structure determination process. Based on this idea, we present three new two-dimensional (2D) NMR experiments, which together provide 15 N, 1 HN, 13 C α , 13 C β , 13 C chemical shifts for amino acid residues which are immediate C-terminal neighbors (i + 1) of residues that are selectively unlabeled. These experiments have high sensitivity and can be acquired rapidly using the methodology of G-matrix Fourier transform (GFT) NMR spectroscopy combined with non-uniform sampling (NUS). This is a first study involving the application of fast NMR methods to proteins samples prepared using a specific labeling scheme. Taken together, this opens up new avenues to using the method of selective unlabeling for rapid resonance assignment of proteins.

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Section: Discussionmentioning

confidence: 79%

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

2017

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“…NMR experiments were recorded at 303 K on a Bruker Avance 800 MHz spectrometer equipped with a 5 mm triple-resonance cryogenic probe and a Varian Inova 600 MHz spectrometer equipped with a triple-resonance probe. Experiments recorded with u-15 N EhCaM included sensitivity-enhanced 2D [ 15 N-1 H]-HSQC using water-flip-back for minimizing water saturation, 3D water-flip-back 15 N-edited TOCSY-HSQC (s m = 80 ms), 15 N-edited NOESY-HSQC (s m = 120 ms), and (3,2)D HNHA (Barnwal et al 2007). The experiments recorded on u-13 C/ 15 N samples were, 3D HNCO, 3D HN(CA)CO, 3D HNCA, 3D CBCANH, and 3D CBCA(CO)NH (Bax and Grzesiek 1993).…”

Section: Methods and Resultsmentioning

confidence: 99%

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

et al. 2008

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“…S3. Further, the proposed ITV-specific (3, 2)D-CB(CACO)NNH experiment has several other advantages, which is characteristic of GFT-NMR Atreya & Szyperski, 2004Barnwal et al, 2007;Kim & Szyperski, 2003;Rout et al, 2010) or RD-NMR (Atreya & Szyperski, 2005;Chandra et al, 2012). For example, for Ubiquitin both cosine-and sine-modulated ITV-specific (3, 2)D-CB(CACO)NNH experiments were recorded with a resolution of 67.5 Hz/pt along the shared ( 15 N/ 13 C) dimension, which is spread over 200 ppm.…”

Section: Peak Patterns In Itv Specific (3 2)d-cb(caco)nnhmentioning

confidence: 99%

Identification of C-Terminal Neighbours of Residues that have only one 1Hβ Attached to 13Cβ: (Ile, Thr and Val)-specific (3,2)D-CB(CACO)NNH Experiment

Chandra¹,

Patel²,

Atreya³

et al. 2014

Proceedings of the Indian National Science Academy

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Sequence specific resonance assignment is the first step in determining the three-dimensional (3D) structure of proteins. Several double and triple-resonance NMR experiments were proposed in this direction for achieving unambiguous assignments. However, these methodologies are non-trivial for proteins with molecular weight beyond 15 kDa. Common problems in this process are spectral overlaps and rapid relaxation rates of the nuclei that result in broad cross peaks. Further, Pro residues, which lack 1 H N , complicate the scenario. This led to identify maximum possible starting points along the polypeptide chain of a given protein to aid in the sequence-specific resonance assignment procedure. In this paper, we propose a fast NMR methodology namely Ile, Thr and Val (ITV) specific (3, 2) D-CB(CACO)NNH, which shows the spectral signatures of the C-terminal sequential neighbors of Ile, Thr and Val residues in highly resolved manner. This aids in the generation of additional starting points along a given polypeptide chain, apart from the known canonical Ala, Gly, Ser and Thr residues, and thus accelerate resonance assignment process significantly. IntroductionAdvent of labeling schemes to produce 13 C-or/and 15 N-enriched proteins paved the way to determine their 3D structures of medium size proteins (upto a M r = 50 kDa) by 3D triple resonance NMR. In this endeavor, 1 H, 13 C and 15 N sequence-specific resonance assignments remain the first step. Till date, several double-and triple-resonance experiments were proposed to carry out these assignments in isotope-labeled proteins. However, these methodologies are non-trivial for proteins with molecular weights even beyond 15 kDa. Common problems in this process are spectral overlaps and rapid relaxation rates of the nuclei that result in broad spectral signatures. Further, Pro residues, which lack 1 H N , complicate the situation. Hence maximum possible number of starting points is required along the polypeptide chain of a given protein to facilitate the sequence-specific resonance assignment procedure.It is a well-known fact that, spectral signatures of Ala, Gly, Ser and Thr residues are easily identifiable residues because of their characteristic 13 C α and 13 C β chemical shifts. The 13 C α chemical shifts of Gly resonate around 45.4±1.8 ppm, which is well separated from the 13 C α chemical shifts of the rest of the residues. On the other hand, the 13 (Atreya et al., 2000;Atreya et al., 2002). Thus, in principle, four tripleresonance experiments HNCACB, CBCA(CO)NH, HNCO and HN(CA)CO, should suffice for the resonance assignment of 1 H N , 15 N, 13 C α , 13 C β and 13 C' spins (Atreya et al., 2000;Atreya et al., 2002). However, in practice, the success rate in any automated resonance assignment procedure turns out to be low, primarily because of incomplete spectral signatures or/and spectral overlap. Hence, the quest has been to identify spectral signatures arising from residues other than Ala, Ser, Thr and Gly. Based on the analysis of BMRB data of 9000 odd protein...

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Rapid measurement of 3J(HN–Hα) and 3J(N–Hβ) coupling constants in polypeptides

Cited by 29 publications

References 23 publications

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

Identification of C-Terminal Neighbours of Residues that have only one <SUP>1</SUP>H<SUP>β</SUP> Attached to <SUP>13</SUP>C<SUP>β</SUP>: (Ile, Thr and Val)-specific (3,2)D-CB(CACO)NNH Experiment

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Rapid measurement of 3J(HN–Hα) and 3J(N–Hβ) coupling constants in polypeptides

Cited by 29 publications

References 23 publications

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

Accelerating NMR-Based Structural Studies of Proteins by Combining Amino Acid Selective Unlabeling and Fast NMR Methods

Sequence specific 1H, 13C and 15N resonance assignments of a calmodulin-like calcium-binding protein from the protozoan parasite Entamoeba histolytica (EhCaM)

Identification of C-Terminal Neighbours of Residues that have only one <SUP>1</SUP>H<SUP>&#946;</SUP> Attached to <SUP>13</SUP>C<SUP>&#946;</SUP>: (Ile, Thr and Val)-specific (3,2)D-CB(CACO)NNH Experiment

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Identification of C-Terminal Neighbours of Residues that have only one <SUP>1</SUP>H<SUP>β</SUP> Attached to <SUP>13</SUP>C<SUP>β</SUP>: (Ile, Thr and Val)-specific (3,2)D-CB(CACO)NNH Experiment