Biological Magnetic Resonance Data Bank

A Repository for Data from NMR Spectroscopy on Proteins, Peptides, Nucleic Acids, and other Biomolecules
Member of WWPDB

Disulfide Formation Predicted with 13C Chemical Shifts
Deepak Sharma and Krishna Rajarathnam

 Department of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology
 5.138 Medical Research Building,
University of Texas Medical Branch,
Galveston, Texas 77555-1055, U.S.A.

Abstract: The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography,  and otherwise must be inferred by chemical methods. In this study we demonstrate that 13C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine 13C Ca and Cb chemical shifts was constructed from the BMRB and Sheffield databases, and  published journals. Statistical analysis indicated that the Cb shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the Ca/Cb chemical shift map.  On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.

Biomol NMR 2000 Oct;18(2):165-71  [PubMed PMID: 11101221]

Ground Rules for deducing the redox state of cysteines in an unknown protein:

Redox state of cysteines in all proteins except those that are paramagnetic can be determined using the following ground rules. To ensure that the redox states are correctly assigned, we suggest that all of the steps are used

Step 1: Ensure that the Ca and Cb assignments of cysteine are correct.

Step 2: If the Cb shift is less than 32.0 ppm or greater than 35.0 ppm, the redox state is assigned to reduced or oxidized, respectively. If the shift lies in the overlap region (32.0 íV 35.0 ppm), the redox state can be assigned using step 3.

Step 3: For proteins with a single cysteine showing a Cb shift in the overlap region, the cysteine must be reduced. For proteins with multiple cysteines, of which one of the Cb shifts lies in overlap region, the oxidation state of this cysteine is established from the Cb shifts of the other cysteines in the protein using Figure 1 and further confirmed from Figure 2. For proteins with multiple cysteines of which more than one lie in the overlap region, the redox state is assigned using Figure 2. In this case, Figure 1 alone cannot unambiguously assign the redox state.

Figures from the article:
Figure one
Figure two
Figure three
Figure four
Figure five

Tabular Data:
Table one
Table two
Table three

Also available is a Microsoft Excel spreadsheet listing data pertaining to the proteins used in this study.

Protein List
this list includes links to the BioMagResBank database and PDB files.

Protein PDB code BMRB code
Tendamistat (a -amylase inhibitor) 1hoe 642
Bovine Pancreas Trypsin Inhibitor (BPTI) 5pti 46
Interleukin 1b 5i1b 1061
Turkey Ovomucoid Domain 3 3sgb 4068
Ribonuclease H 2rn2 1657
protein kinase c-gamma phorbol bindingdomain h102, c132, c135, c151, h140, c115, c118, c143 .
FK506 binding protein 1fkf .
Horse ferrocytochrome 2pcb .
Flavodoxin 1flv 1580.
Interleukin 4 1rcb 4094
Cyclophilin 2rma 2208    .
Delta NZF1-3  . .
Human profilin 2btf .
Chicken cystatin 1cew .
Cow tyrosine phosphatase 1pnt .
Human Gcsf 1rhg .
Phosphoglycerate Kinase 1php .
Transforming Growth Factor 2tgi .
Cytochrome C2 3c2c, 1c2n .
Ras p21 4q21 .
Recoverin 1rec .
Ribonuclease T1 9rnt .
Phospholipase A2 2bpp .
Troponin C 1top .
HIV Zinc Fingerlike Domain . 1640
reduced apo-S100beta 1qlk, 1sym 4001
Recombinant Poplar Plastocyanin 5pcy 4019
Immunodominant Region of Protein G ofBovine Respiratory Syncytial Virus 1brv 4020
Human Carbonic Anhydrase I 1azm 4022
Sex-lethal protein 3sxl 4029
DNA-Binding Domain of a Human PapillomavirusE2 1dhm 4035
Apokedarcidin 1akp 4036
Component IV Glycera Dibranchiata MonomericHemoglobin-CO 1vrf 4038
C-Terminal Single-Stranded DNA-BindingDomain of E. Coli Topoisomerase I 1yua 4045
Drosophila Heat Shock Factor 1hks 4046
Coiled Coil trimerization domain of ChickenCartilage Matrix Protein' 1aq5 4055
Oncostatin M . 4063
Brazzein 1brz 4067
RNA-1_Modulator_Protein 1rpr 4072
Oxidized Human Ferredoxin . 4073
A-domain_HMG1 . 4079
Human Interferon alpha-2a 1itf 4081
RNA Binding Domain of Mammalian nhRNPA1 Protein . 4084
Tumor Suppressor p16ink4a . 4086
DNA-Binding Domain of the BPV-1 E2 Protein 1dbd 4087
Peptide Deformylase Catalytic Core (Residues1 - 147) 1def 4089
Basic Fibroblast Growth Factor 1bla 4091
Core binding factor (CBF) 2jhb 4092
Liver Fatty Acid-Binding Protein 1lfo 4098
S100B(bb)with a Peptide Derived from p53 . 4099
Mrf-2 DNA-binding Domain 1bmy 4100
Fusarium Solani Pisi Cutinase 1oxm 4101
Metallo-beta-lactamase 1bmi, 2bmi 4102
reduced s100B 1cfp 4105
double stranded RNA binding domain ofPKR 1qu6 4110
Intimin cell adhesion domain 1inm 4111
Vaccinia Glutaredoxin-1(Fully ReducedForm) . 4113
colicin E9 immunity protein Im9 boundto colicin E9 DNase domain 1imq 4115
Immunity protein Im9 1imp 4116
human Elongation Factor-1beta . 4117
1 Homeodomain 1bw5 4121
DNA-binding Domain of SV40 T Antigen 1tbd 4127
Cyanobacterial Transcriptional Factor 1smt 4128
Human UBC9 . 4132
Denatured LysN 1krt 4134
E.coli Multidrug Resistance Protein . 4136
EH1 domain of mouse Eps15 1qjt 4140
Gallium-Substituted Putidaredoxin Mutant:GaPdx C85S 1gpx .
Adenylate kinase 2eck 4152
Oxidized Putidaredoxin 1pdx, 1put, 1gpx 4154
Tn916 integrase subunit 1 2bb8 4160
Neural Cell Adhesion Molecule Module 1 2ncm 4162
antibody Fv - V3 antigen 2bfv 4171
Structure and Asn-Pro-Phe Binding Pocketof the Eps15 Homology Domain 1eh2 4184
Human Cellular Retinoic Acid Binding Protein-TypeII 1cbq, 2cbs, 3cbs, 1cbs 4186
Adenylate kinase/AP5A complex 2eck, 4ake 4193
Ets-1 Pointed 1bqv 4205
MinE subunit 1 . 4237
Motile Major Sperm Protein 3msp, 1msp 4242
RPA-binding Domain of the Human Repairfactor xpa 1xpa, 1d4u 4249
Human Translation Initiation Factor eIF1 2if1 4255
Ribonuclease Sa 1ay7, 1rgh, 1rgg, 1rge 4259
dNumb PTB Domain Complexed with a PhosphotyrosinePeptide 2nmb 4263
Chlamydomonas Outer Arm Dynein . 4265
HNGAL (Human Neutrophil Gelatinase-AssociatedLipocalin) in its apo form 1ngl 4267
Methyl-CpG-binding Protein MeCP2. 1qk9 4280
Apo-S100A1 . 4285
EH2 Domain of Mouse Eps15 . 4288
Protein P14a 1cfe 4301
Domain of Protein Disulfide Isomerase (pdbexists for frg A domain B) 4302
Structure of the HTLV-I Capsid Protein 1qrj 4311
Histone Acetyltransferase hsP/CAF 1b91 4312
E-2/E-2' protein [Klebsiella oxytoca] . 4313
NS1(1-73) 1AIL, 1NS1 4317
Reduced Escherichia coli Glutaredoxin2 1de1, 1de2 4318
N-terminal Inhibitory Domain of HumanTissue Inhibitor of Metalloproteinases-1 1d2b, 1uea 4327
Human Rad51(1-114) 1b22  4328
Streptomyces Subtilisin Inhibitor 3ssi, 2sic, 2tld 4331
ARID Domain of Dead-Ringer Protein 1c20 4334
Calerythrin . 4335
Lipoate Containing H-protein of the GlycineDecarboxylase Complex 1hpc, 1htp 4336
Fv Fragment of the Catalytic AntibodyNPN43C9 with Bound p-nitrophenol 43ca, 43c9 4349
C-terminal Domain of MutY . 4353
Pbx1 TALE Homeodomain Protein 1b72 4357
C2B-domain of Rabphilin 3 3rpb 4360
Onconase/P30 Protein 1onc 4371
N-domain of VAT 1cz4, 1cz5 4376
Human Prion Protein Domain (23-230) 1qm2, 1qm3,1qm0 4402
Amino-Terminal Extracellular Domain ofEpithelial Cadherin 1edh, 1suh 4380
Rous Sarcoma Virus Capsid Protein 1d1d 4384
CX3C Chemokine Domain of Fractalkine 1b2t 4397
Galpha Interacting Protein 1cmz 4407
N-terminal Domain of Saccharomyces cerevisiaeRNase HI 1qhk 4424
apo-biotinyl domain from acetyl coenzymeA carboxylase   3bdo, 1a6x, 1bdo 4425
Methane Monooxygenase Regulatory ProteinB 1ckv 4431
EGF module pair from the Plasmodium falciparummerozoite surface protein 1  1cej 4437
native hen egg white lysozyme 1lsg, 1lyz 4562
APAF-1 CARD 1cy5r, 1cww 4661
VEGF . .
reduced E.Coli DsbA . .
Oxidised E.Coli DsbA . .
reduced E.Coli thioredoxin . .
oxidised E.Coli thioredoxin . .
w-conotoxin MVIIA . .
reduced human thioredoxin . .
oxidised human thioredoxin . .