Structures of N‐termini of helices in proteins

Doig, Andrew J.; MacArthur, Malcolm W.; Stapley, Benjamin J.; Thornton, Janet M.

doi:10.1002/pro.5560060117

Cited by 114 publications

(107 citation statements)

References 35 publications

(28 reference statements)

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“…Nonetheless, the N-Cap Cys motif might serve as a useful SNO-site predictor: N termini of helices in proteins are often solvent-exposed (55), and charge stabilization by the helix dipole may lower the pK a of the N-Cap Cys by approximately two pH units (56,57) [a potentially greater contribution to pK a than a proximal basic (e.g., His) residue (58)]. Notably, the catalytic cysteines of rhodanese/Cdc25 phosphatases and PTPs are near the cationic side of a helix (Fig.…”

Section: Discussionmentioning

confidence: 99%

A protein microarray-based analysis of S -nitrosylation

Foster

Forrester

Stamler

2009

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

105

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The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of ␣-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.cysteine ͉ nitric oxide ͉ S-nitrosothiol ͉ thiol P rotein S-nitrosylation underlies much of the physiological signaling by both nitric oxide (NO) and endogenous Snitrosothiols, and both hypo-and hyper-S-nitrosylation have been causally implicated in disease (1, 2). Formally, Snitrosylation occurs either via an oxidative reaction of NO and Cys thiol, in the presence of an electron acceptor (e.g., transition metal or O 2 ), or by the transfer of NO ϩ (transnitros(yl)ation) from donor S-nitrosothiol (SNO) to acceptor Cys thiol (1). These reactions may be enzyme-catalyzed or otherwise facilitated. For example, hemoglobin and ceruloplasmin support metaldependent S-nitrosylation (3-5) whereas S-nitroso-hemoglobin (SNO-hemoglobin) and SNO-thioredoxin can transnitrosylate proteins with which they interact directly (anion exchanger 1 and caspase-3, respectively) (6, 7). Transnitrosylation is also implicated in protein S-nitrosylation coupled to NO synthase activity (8-10) and involves the intermediacy of the endogenous lowmass SNO, S-nitrosoglutathione (GSNO), as evidenced by elevated levels of SNO-proteins in mice lacking the GSNO metabolizing enzyme, GSNO reductase (GSNOR) (8-11). GSNOR deletion also increases S(NO)-mediated protein S-nitrosylation and cytostasis in yeast (11,12), and decreases virulence of several pathogens (13, 14), which suggests that transnitrosylation is an important mediator of nitrosative stress. In addition, metabolism of GSNO to S-nitrosocyteinylglycine and S-nitrosocysteine (CysNO) and CysNO uptake via l-amino...

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Section: Discussionmentioning

confidence: 99%

A protein microarray-based analysis of S -nitrosylation

Foster

Forrester

Stamler

2009

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

105

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“…Helix capping. DTBS has eight -helices and most of them have an N-cap residue with high N-capping propensity (Doig et al, 1997), standard geometry and hydrogen-bond pattern. However, the conformation of the N-cap residue Glu94 of helix 6 is rather unusual.…”

Section: Hydrogen Bonds In Dtbsmentioning

confidence: 99%

Structure of dethiobiotin synthetase at 0.97 Å resolution

Sandalova¹,

Schneider²,

Käck³

et al. 1999

Acta Crystallogr D Biol Crystallogr

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The crystal structure of the 224-residue protein dethiobiotin synthetase from Escherichia coli has been re®ned using X-ray diffraction data at 0.97 A Ê resolution at 100 K. The model, consisting of 4143 protein atoms including 1859 H atoms and 436 solvent sites, was re®ned to a ®nal R factor of 11.6% for all re¯ections, and has an estimated mean standard uncertainty for the atomic positions of 0.022 A Ê , derived from inversion of the blocked matrix. The structure was re®ned with a full anisotropic model for the atomic displacement parameters using SHELX97. Stereochemical restraints were applied throughout the re®nement. In the last cycles, the planarity of the peptide bonds was not restrained, resulting in a mean 3 value of 179.6. Analysis of the most anisotropic regions of the protein shows that they form four clusters of residues. Alternate conformations for the side chains of 15 residues and for the main-chain atoms of six residues from three loops were included in the model. An analysis of CÐHÁ Á ÁO hydrogen bonds shows that such interactions occur rather frequently in DTBS; in total, 16 such hydrogen bonds were found. In the central -sheet, 13 CÐHÁ Á ÁO bonds between carbonyl O and C H atoms were found. Other interactions of this type involve main-chain±side-chain and side-chain±side-chain CÐHÁ Á ÁO bonds. The model includes 436 water sites, of which 233 molecules form the ®rst hydration shell. Analysis of the protein±solvent interactions shows that about one third of the accessible surface of the enzyme is not covered by ordered solvent. No difference in propensity for ordered solvent close to hydrophilic or hydrophobic surface areas was found. The comparison of the 100 K structure with the structure of the enzyme determined at room temperature shows several regions with different conformation, including areas in the active site, suggesting that structural transitions can occur during¯ash freezing. This observation questions one of the basic assumptions in the analysis of enzymatic reaction mechanisms using cryocrystallography.

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“…it is the negatively charged side that is exposed and, at the N-terminus, it is the positively charged side. This is why the N-termini of a-helices often function as binding sites for inorganic anions (Hol et al, 1978 Doig et al, 1997). Earlier work (Hol et al.…”

Section: Relevance To Aspects Of Proteinsmentioning

confidence: 99%

The partial charge of the nitrogen atom in peptide bonds

1997

Protein Science

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A majority of the standard texts dealing with proteins portray the peptide link as a mixture of two resonance forms, in one of which the nitrogen atom has a positive charge. As a consequence, it is often believed that the nitrogen atom has a net positive charge. This is in apparent contradiction with the partial negative charge on the nitrogen that is used in force fields for molecular modeling. However, charges on resonance forms are best regarded as formal rather than actual charges and current evidence clearly favors a net negative charge for the nitrogen atom. In the course of the discussion, new ideas about the electronic structure of amides and the peptide bond are presented.Keywords: a-helix; amides; electrostatics; hydrogen bonds; proteins; quantum mechanics This article begins with a listing of some estimates of the partial charges of the atoms of amides. The planarity of amides is typically explained by portraying them as two resonance forms, one of which has a nitrogen atom with a formal positive charge. However, it is chemically unsatisfactory for the nitrogen atom to have a net partial positive charge, and the reasons for this are given. The strongest evidence that the nitrogen is negative rather than positive comes from quantum mechanics. Some recent work on amides and its implications for the electronic structure of the and T orbitals of the CONH group are discussed in some detail. The effect of hydrogen bonding is then considered and, finally, the relevance of these insights for a-helices is addressed. Point charges on individual atoms:The resonance method used commonly to represent the peptide bond as in Figure 1 is often taken to imply that the nitrogen has a partial positive charge. This conflicts with the partial charges used widely in molecular mechanics force fields for modeling biological molecules. A set of calculated partial charges has been collected (Rogers, 1986), and a

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Structures of N‐termini of helices in proteins

Cited by 114 publications

References 35 publications

A protein microarray-based analysis of S -nitrosylation

A protein microarray-based analysis of S -nitrosylation

Structure of dethiobiotin synthetase at 0.97 Å resolution

The partial charge of the nitrogen atom in peptide bonds

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