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1.
Tran‐Chin Yang Jennifer Rendell Wayne Gulliver Valerie Booth 《Journal of peptide science》2009,15(12):818-823
The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure‐inducing conditions: 40% hexafluoro‐2‐propanol aqueous solution at 5 °C. Under these conditions it was possible to detect medium‐range NOEs for the first time for this peptide. This allowed a much better‐defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4–8 of Peptide T take on a well‐defined structure with a heavy atom RMSD of 0.78 Å. The structure is stabilized by hydrogen bonding to side‐chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic γ‐turn. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
2.
Fred M. Kaplan Curtis H. Kugel III Neda Dadpey Yongping Shao Ethan V. Abel Andrew E. Aplin 《The Journal of biological chemistry》2012,287(50):41797-41807
ERK1/2 signaling is frequently dysregulated in tumors through BRAF mutation. Targeting mutant BRAF with vemurafenib frequently elicits therapeutic responses; however, durable effects are often limited by ERK1/2 pathway reactivation via poorly defined mechanisms. We generated mutant BRAFV600E melanoma cells that exhibit resistance to PLX4720, the tool compound for vemurafenib, that co-expressed mutant (Q61K) NRAS. In these BRAFV600E/NRASQ61K co-expressing cells, re-activation of the ERK1/2 pathway during PLX4720 treatment was dependent on NRAS. Expression of mutant NRAS in parental BRAFV600 cells was sufficient to by-pass PLX4720 effects on ERK1/2 signaling, entry into S phase and susceptibility to apoptosis in a manner dependent on the RAF binding site in NRAS. ERK1/2 activation in BRAFV600E/NRASQ61K cells required CRAF only in the presence of PLX4720, indicating a switch in RAF isoform requirement. Both ERK1/2 activation and resistance to apoptosis of BRAFV600E/NRASQ61K cells in the presence of PLX4720 was modulated by SHOC-2/Sur-8 expression, a RAS-RAF scaffold protein. These data show that NRAS mutations confer resistance to RAF inhibitors in mutant BRAF cells and alter RAF isoform and scaffold molecule requirements to re-activate the ERK1/2 pathway. 相似文献
3.
Olivier Julien Pascal Mercier Melissa L. Crane Brian D. Sykes 《Protein science : a publication of the Protein Society》2009,18(6):1165-1174
The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N‐domain of cardiac troponin C (F77W‐cNTnC) to study the domain orientation of troponin C in muscle fibers using solid‐state NMR. In our previous study, we determined the NMR structure of calcium‐saturated mutant F77W‐V82A‐cNTnC in the presence of 19% 2,2,2‐trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W‐cNTnC in presence of its biological binding partner troponin I peptide (cTnI144–163) and in the absence of TFE. We have also characterized a panel of six F77W‐cNTnC structures in the presence and absence TFE, cTnI144–163, and the extra mutation V82A, and used 19F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents. 相似文献
4.
Takashima H Tamaoki H Teno N Nishi Y Uchiyama S Fukui K Kobayashi Y 《Biochemistry》2004,43(44):13932-13936
Human endothelin-1 (ET-1) is a potent cardiovascular bioactive peptide. Its activity is based on the C-terminal residues, e.g., Trp 21 in particular. Recently, we reported an NMR solution structure of ET-1, which has a C-terminal hydrophobic core around Tyr 13. This C-terminal conformation does not agree with a previously reported X-ray crystal structure. To clarify the discrepancy, we performed photo-CIDNP NMR in combination with MALDI-TOF MS. The photo-CIDNP results revealed that the Tyr 13 aromatic ring is concealed in a hydrophobic interaction. MALDI-TOF MS experiments showed this is an intramolecular interaction in monomeric form, which is also supported by sedimentation analysis and two-dimensional NMR cross-peak line shapes. Thus, we confirmed the intramolecular hydrophobic core around Tyr 13 in aqueous solution, which agrees with the solution structure. The C-terminal conformational discrepancy between the solution and crystal was caused by the intermolecular hydrogen bond between Tyr 13 of one molecule and Asp 8 of the other in a dimer-like formation of crystalline ET-1. On the other hand, we indicated that endothelin-3, another isoform of the endothelin, has an apparent self-association equilibrium under the same condition in which three tyrosines participate. 相似文献
5.
Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform of FAIM is expressed in all tissue types, while an alternatively spliced long isoform is specifically expressed in the brain. Here, the short isoform is shown to consist of two independently folding domains in contact with each other. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded β-sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N-terminal domain and the C-terminal domain. 相似文献
6.
Shuyan Xiao Yuxia Wang Lei Yang Haiyan Qi Chunyu Wang Fei Li 《Journal of peptide science》2010,16(5):249-255
The structure and self‐assembly of the peptide corresponding to the third transmembrane domain (TMD3) of Slc11a1 and its E139A mutant are studied in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) aqueous solution by NMR and CD experiments. Slc11a1 is an integral membrane protein with 12 putative TMDs and functions as a pH‐coupled divalent metal cation transporter. Glu139 of Slc11a1 is highly conserved within predicted TMD3 of the Slc11 protein family and function‐associated. Here, we provide the first direct experimental evidence for the structural features of two 24‐residue peptides corresponding to TMD3 of Slc11a1 and its E139A mutant in 60% HFIP‐d2 aqueous solution using CD and NMR spectroscopies. Our study shows that the membrane‐spanning peptide folds as a typical amphipathic α‐helix structure from Ile5 to Met20 with hydrophilic residues Glu12 (Glu139 in Slc11a1) and Asp19 lying on the same side of the helix. The substitution of Glu139 by an alanine residue has little effect on the structure of the peptide, but increases hydrophobicity and facilitates self‐assembly of the peptide. Although the wildtype peptide is monomeric in HFIP aqueous solution, the E139A mutant forms a dimer. The increase in hydrophobicity of the membrane‐spanning peptide and/or change in the interactions between transmembrane segments induced by E139A mutation may affect the metal ion transport of the protein. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
7.
Solution NMR structure of zinc finger 4 and 5 from human INSM1, an essential regulator of neuroendocrine differentiation
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Jiang Zhu Huapu Wang Theresa A. Ramelot Michael A. Kennedy Rui Hu Xiali Yue Maili Liu Yunhuang Yang 《Proteins》2017,85(5):957-962
Human INSM1 containing five C‐terminal C2H2‐type zinc fingers (ZFs), is a key regulator of neuroendocrine development. Previous research reported that full‐length INSM1 containing all five ZFs recognized a consensus DNA sequence. Structure elucidation of human INSM1 ZFs is currently insufficient to understand the DNA binding mechanism. Herein, we present the solution NMR structure of ZF4‐5, in which the two ZFs adopt a head‐to‐tail arrangement and each ZF features a canonical ββα fold. NMR titrations and isothermal titration calorimetry experiments showed that ZF4‐5 binds weakly to the consensus DNA sequence. Proteins 2017; 85:957–962. © 2016 Wiley Periodicals, Inc. 相似文献
8.
Elisa Bertoli Marco Giavarra Maria Grazia Vitale Alessandro Marco Minisini 《Pigment cell & melanoma research》2019,32(6):744-752
Neuroblastoma rat sarcoma (NRAS) mutation, occurring in about 20%–30% of cutaneous melanomas, leads to activation of RAS‐RAF‐MAPK cascade and represents a clear distinct clinicopathological entity in melanoma. In contrast with BRAF mutant melanoma, no specific target therapies are available outside the setting of clinical trials. In the field of immunoncology, the predictive role of NRAS mutation with respect to checkpoint inhibitors treatment has not clearly established and deserves further investigation. At present, the standard treatment is the same as for BRAF wild type melanoma. Ongoing trials are exploring novel combination strategies among patients with advanced NRAS mutant melanoma. 相似文献
9.
Comparison of NMR and crystal structures of membrane proteins and computational refinement to improve model quality
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Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is “which structure is most biologically relevant?” Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X‐ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 Å in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo‐chemical correctness, and are more tightly packed. After quantifying these differences, we used high‐resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins. 相似文献
10.
Kazuhide Miyamoto Kayo Togiya Ryo Kitahara Kazuyuki Akasaka Yoshihiro Kuroda 《Journal of peptide science》2010,16(4):165-170
The synthetic peptide fragment (LC5: LRCRNEKKRHRAVRLIFTI) inhibits human immunodeficiency virus type 1 (HIV‐1) infection of MT‐4 cells. In this study, the solution structure of LC5 in SDS micelles was elucidated by using the standard 1H two‐dimensional NMR spectroscopic method along with circular dichroism and fluorescence quenching. The peptide adopts a helical structure in the C‐terminal region (residues 13–16), whereas the N‐terminal part remains unstructured. The importance of Phe17 in maintaining the structure of LC5 was demonstrated by replacing Phe17 with Ala, which resulted in the dramatic conformational change of LC5. The solution structure of LC5 elucidated in the present work provides a basis for further study of the mechanism of the inhibition of HIV‐1 infection. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
11.
Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma
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Michal Alon Rafi Emmanuel Nouar Qutob Anna Bakhman Victoria Peshti Alexandra Brodezki David Bassan Mickey Kosloff Yardena Samuels 《Pigment cell & melanoma research》2018,31(5):641-648
The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor‐intensive. To this end, we present a polyadenylation signal‐trap construct for N’‐tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c‐CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants. 相似文献
12.
Huang KY Amodeo GA Tong L McDermott A 《Protein science : a publication of the Protein Society》2011,20(3):630-639
A new crystal structure of human ubiquitin is reported at 1.8 Å resolution. Compared with the other known crystal structure or the solution NMR structure of monomeric human ubiquitin, this new structure is similar in its overall fold but differs with respect to the conformation of the backbone in a surface‐exposed region. The conformation reported here resembles conformations previously seen in complex with deubiquinating enzymes, wherein the Asp52/Gly53 main chain and Glu24 side chain move. This movement exposes the backbone carbonyl of Asp52 to the exterior of the molecule, making it possible to engage in hydrogen‐bond contacts with neighboring molecules, rather than in an internal hydrogen bond with the backbone of Glu24. This particular crystal form of ubiquitin has been used in a large number of solid state NMR studies. The structure described here elucidates the origin of many of the chemical shift differences comparing solution and solid state studies. 相似文献
13.
The membrane localization domains of two distinct bacterial toxins form a 4‐helix‐bundle in solution
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Grant S. Hisao Michael C. Brothers Mengfei Ho Brenda A. Wilson Chad M. Rienstra 《Protein science : a publication of the Protein Society》2017,26(3):497-504
Membrane localization domain (MLD) was first proposed for a 4‐helix‐bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1‐specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats‐in‐toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1‐C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4‐helix‐bundle structure in solution. 相似文献
14.
Ai‐Xin Song Chen‐Jie Zhou Xiao Guan Kong‐Hung Sze Hong‐Yu Hu 《Protein science : a publication of the Protein Society》2010,19(5):1104-1109
DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells. 相似文献
15.
This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) 1H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
16.
Janosch Hennig Michael Sattler 《Protein science : a publication of the Protein Society》2014,23(6):669-682
Structural biology provides essential information for elucidating molecular mechanisms that underlie biological function. Advances in hardware, sample preparation, experimental methods, and computational approaches now enable structural analysis of protein complexes with increasing complexity that more closely represent biologically entities in the cellular environment. Integrated multidisciplinary approaches are required to overcome limitations of individual methods and take advantage of complementary aspects provided by different structural biology techniques. Although X‐ray crystallography remains the method of choice for structural analysis of large complexes, crystallization of flexible systems is often difficult and does typically not provide insights into conformational dynamics present in solution. Nuclear magnetic resonance spectroscopy (NMR) is well‐suited to study dynamics at picosecond to second time scales, and to map binding interfaces even of large systems at residue resolution but suffers from poor sensitivity with increasing molecular weight. Small angle scattering (SAS) methods provide low resolution information in solution and can characterize dynamics and conformational equilibria complementary to crystallography and NMR. The combination of NMR, crystallography, and SAS is, thus, very useful for analysis of the structure and conformational dynamics of (large) protein complexes in solution. In high molecular weight systems, where NMR data are often sparse, SAS provides additional structural information and can differentiate between NMR‐derived models. Scattering data can also validate the solution conformation of a crystal structure and indicate the presence of conformational equilibria. Here, we review current state‐of‐the‐art approaches for combining NMR, crystallography, and SAS data to characterize protein complexes in solution. 相似文献
17.
Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS‐mutant and wild‐type melanoma
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Alain P. Algazi Rosaura Esteve‐Puig Adi Nosrati Brian Hinds Adele Hobbs‐Muthukumar Prachi Nandoskar Susana Ortiz‐Urda Paul B. Chapman Adil Daud 《Pigment cell & melanoma research》2018,31(1):110-114
Aberrant MAPK and PI3K pathway signaling may drive the malignant phenotype in NRAS‐mutant and BRAFWT NRASWT metastatic melanoma. To target these pathways, NRAS‐mutant and BRAFWT NRASWT patients received oral trametinib at 1.5 mg daily and GSK2141795 at 50 mg daily in a two‐cohort Simon two‐stage design. Participants had adequate end‐organ function and no more than two prior treatment regimens. Imaging assessments were performed at 8‐week intervals. A total of 10 NRAS‐mutant and 10 BRAFWT NRASWT patients were enrolled. No objective responses were noted in either cohort. The median PFS and OS were 2.3 and 4.0 months in the NRAS‐mutant cohort and 2.8 and 3.5 months in the wild‐type cohort. Grade 3 and grade 4 adverse events, primarily rash, were observed in 25% of patients. We conclude that the combination of trametinib and GSK2141795 does not have significant clinical activity in NRAS‐mutant or BRAFWT NRASWT melanoma. 相似文献
18.
Hao Huang Hiroaki Ishida Hans J. Vogel 《Protein science : a publication of the Protein Society》2010,19(3):475-485
Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca2+. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg2+ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg2+ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg2+ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca2+ form. We have used the NMR backbone residual dipolar coupling restraints 1DNH, 1DCαHα, and 1DC′Cα to determine the solution structure of the N‐terminal domain of Mg2+‐sCaM4 (Mg2+‐sCaM4‐NT). Compared with the known structure of Ca2+‐sCaM4, the structure of the Mg2+‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg2+‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg2+‐sCaM4 does not bind to Ca2+‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg2+‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells. 相似文献
19.
Yaoqing Diana Liu Robert Y.‐T. Chou Thomas M. Dillon Leszek Poppe Chris Spahr Stone D. H. Shi Gregory C. Flynn 《Protein science : a publication of the Protein Society》2014,23(12):1753-1764
Human IgG2 consists of disulfide‐mediated structural isoforms, classified by the number of Fab arms disulfide‐linked to the heavy chain hinge. In the IgG2‐B isoform, both Fab arms are linked to the hinge region, and in IgG2‐A, neither Fab arm are linked to the hinge. IgG2‐A/B is a hybrid between these two forms, with only one Fab arm disulfide‐linked to the hinge. Within each of these isoform types are subtypes, with subtle disulfide‐linkage differences. Here we explored the structural basis for the A1 and A2 isoform subtypes. Whereas A1 isoform converts into the A/B and B isoforms under mild redox conditions, A2 does not. Characterization of the disulfide connectivities of A2 isoform revealed a similar structure to A1 isoform, with parallel inter heavy chain disulfide linkages in the hinge region. However, the hinge disulfides in A2 isoform were resistant to reduction under conditions where A1 isoform hinge disulfides became reduced and they required thermal treatment (>55°C) to obtain thiol‐dependent disulfide reduction. Structural analysis of the hinge region indicated that the protected disulfides were restricted to cysteines 219 and 220 of the upper hinge. Disruption of the upper hinge through insertion mutagenesis eliminated A2 isoform behavior. 1H NMR studies showed that the A1 isoform Fc glycan was more dynamic than that on A2 isoform and showed some other conformational differences. Results point to an IgG2‐A2 upper hinge region that is more akin to the interior of a globular protein than the flexible hinge region expected on an IgG. 相似文献
20.
Tao Huang Jess Li R. Andrew Byrd 《Protein science : a publication of the Protein Society》2014,23(5):662-667
Lysine‐free ubiquitin (K0‐Ub) is commonly used to study the ubiquitin‐signaling pathway, where it is assumed to have the same structure and function as wild‐type ubiquitin (wt‐Ub). However, the K0‐Ub 15N heteronuclear single quantum correlation NMR spectrum differs significantly from wt‐Ub and the melting temperature is depressed by 19°C, raising the question of the structural integrity and equivalence to wt‐Ub. The three‐dimensional structure of K0‐Ub was determined by solution NMR, using chemical shift and residual dipolar coupling data. K0‐Ub adopts the same backbone structure as wt‐Ub, and all significant chemical shifts can be related to interactions impacted by the K to R mutations. 相似文献