Disulfide Bond Isomerization in Basic Pancreatic Trypsin Inhibitor:  Multisite Chemical Exchange Quantified by CPMG Relaxation Dispersion and Chemical Shift Modeling

Grey, Michael J.; Wang, Chunyu; Palmer, Arthur G.

doi:10.1021/ja0367389

Cited by 146 publications

(225 citation statements)

References 85 publications

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“…The protonation state of ionizable groups corresponds to neutral pH, with the net protein charge neutralized by six chloride ions. A subset of 1,048,349 frames with 0.25-ns spacing, corresponding to a 0.262-ms-long trajectory, was extracted by requiring that the 14-38 disulfide bond is in the experimentally dominant M1 conformation (54). Further details can be found in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

How amide hydrogens exchange in native proteins

Persson

Halle

2015

Proc. Natl. Acad. Sci. U.S.A.

127

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Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.protein dynamics | hydration | proton transfer | MD simulation | BPTI B efore the tightly packed and densely H-bonded structure of globular proteins had been established, Hvidt and Linderstrøm-Lang (1) showed that all backbone amide hydrogens of insulin exchange with water hydrogens, implying that all parts of the polypeptide backbone are, at least transiently, exposed to solvent. In the following 60 y, hydrogen exchange (HX), usually monitored by NMR spectroscopy (2) or mass spectrometry (3), has been widely used to study protein folding and stability (4-10), structure (11, 12), flexibility and dynamics (13-15), and solvent accessibility and binding (16,17), often with single-residue resolution. However, because the exchange mechanism is unclear, HX data from proteins can, at best, be interpreted qualitatively (18)(19)(20)(21)(22)(23)(24)(25).Under most conditions, amide HX is catalyzed by hydroxide ions (26, 27) at a rate that is influenced by inductive and steric effects from adjacent side chains (28). For unstructured peptides, HX is a slow process simply because the hydroxide concentration is low. For example, at 25°C and pH 4, HX occurs on a time scale of minutes. Under similar conditions, amides buried in globular proteins exchange on a wide range of time scales, extending up to centuries. HX can only occur if the amide is exposed to solvent, so conformational fluctuations must be an integral part of the HX mechanism (18).Under sufficiently destabilizing conditions HX occurs from the denatured-state ensemble, but under native conditions few amides exchange by such global unfolding (9, 29-31). For example, in bovine pancreatic trypsin inhibitor (BPTI), 8 amides in the core β-sheet exchange by global unfolding under native conditions (7, 32), whereas the remaining 45 amides require less extensive conforma...

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Section: Methodsmentioning

confidence: 99%

How amide hydrogens exchange in native proteins

Persson

Halle

2015

Proc. Natl. Acad. Sci. U.S.A.

127

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“…In very favorable cases a three state model can be used without additional constraints such as addition of ligands or change of temperature (Meinhold & Wright, 2011). There exist a few cases in which three-state models have successfully been applied to proteins undergoing exchange, such as the study by Grey et al (2003) on the multiple states induced by disulfide bond isomerization in the basic pancreatic trypsin inhibitor, and the calculation of structural ensembles of folding intermediates for two Fyn SH3 domain variants reported by Korzhnev et al (2004). It should be noted that the three-state model in Korzhnev et al (2004), was applied under the assumption that (i) the chemical shift differences between exchanging states are temperature-independent, and (ii) the temperature dependence of the rate constants obeys standard transition-state theory (Fersht, 2000).…”

Section: Exchange Modelsmentioning

confidence: 99%

Protein dynamics and function from solution state NMR spectroscopy

Kovermann

Rogne

Wolf‐Watz

2016

Quart. Rev. Biophys.

148

122

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Abstract. It is well-established that dynamics are central to protein function; their importance is implicitly acknowledged in the principles of the Monod, Wyman and Changeux model of binding cooperativity, which was originally proposed in 1965. Nowadays the concept of protein dynamics is formulated in terms of the energy landscape theory, which can be used to understand protein folding and conformational changes in proteins. Because protein dynamics are so important, a key to understanding protein function at the molecular level is to design experiments that allow their quantitative analysis. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited for this purpose because major advances in theory, hardware, and experimental methods have made it possible to characterize protein dynamics at an unprecedented level of detail. Unique features of NMR include the ability to quantify dynamics (i) under equilibrium conditions without external perturbations, (ii) using many probes simultaneously, and (iii) over large time intervals. Here we review NMR techniques for quantifying protein dynamics on fast (ps-ns), slow (μs-ms), and very slow (s-min) time scales. These techniques are discussed with reference to some major discoveries in protein science that have been made possible by NMR spectroscopy.

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“…In addition, increased transverse 13 C relaxation rates were observed for the C α resonances of Cys14 and Cys38 [9]. Furthermore, it was reported that isomerization of the Cys14 side chain between χ 1 rotamers is faster than the corresponding Cys38 isomerization [10].…”

Section: Introductionmentioning

confidence: 94%

“…Cys14 of BPTI is adjacent to the P1 position, Lys15, and forms a disulfide bond with Cys38, while the corresponding P1 position in BF9 is Asn17 and the corresponding disulfide bond is Cys16-Cys40 based on sequence alignment of BPTI and BF9 [5]. Due to isomerization of the Cys14-Cys38 disulfide bond, two conformational isomers with different chirality were observed in BPTI in the NMR spectra [9,10]. The population of the two isomers is temperature-dependent.…”

Section: Introductionmentioning

confidence: 99%

NMR Solution Structure of a Chymotrypsin Inhibitor from the Taiwan Cobra Naja naja atra

et al. 2013

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Abstract:The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 3 10 -helix, one α-helix and one double-stranded antiparallel β-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI OPEN ACCESSMolecules 2013, 18 8907 might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14-Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14-Cys38 disulfide bond.

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Disulfide Bond Isomerization in Basic Pancreatic Trypsin Inhibitor: Multisite Chemical Exchange Quantified by CPMG Relaxation Dispersion and Chemical Shift Modeling

Cited by 146 publications

References 85 publications

How amide hydrogens exchange in native proteins

How amide hydrogens exchange in native proteins

Protein dynamics and function from solution state NMR spectroscopy

NMR Solution Structure of a Chymotrypsin Inhibitor from the Taiwan Cobra Naja naja atra

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