Exposure of tryptophanyl residues and protein dynamics

Eftink, Maurice R.; Ghiron, Camillo A.

doi:10.1021/bi00644a024

Cited by 297 publications

(206 citation statements)

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“…The spectra were normalized and averaged (n ϭ 11 for Na V 1.5 EF in buffer, and n ϭ 4 under denaturing conditions). The black square indicates the emission maximum for Na V 1. the tryptophan is not accessible and 17.5 for free tryptophan (24). The K SV we found for the native protein thus describes a tryptophan that is partially accessible, consistent with fluorescence emission maximum and the structural model.…”

Section: Thermodynamic Cycle Analysis Is Consistent With Predictedsupporting

confidence: 84%

“…Biochemical Evidence for an H1/H4 Hydrophobic InterfaceThe predicted EF-hand pair in the Na V 1.5 COOH terminus contains a single tryptophan, Trp 1798 , which is important not only because our model predicts it to be integral to the hydrophobic interface but also because its intrinsic fluorescence can be used to probe and report the nature of the environment in which it is located (13,24,25). We thus prepared a GST fusion protein consisting of residues predicted to form the EF-hand pair (residues 1786 -1863) and carried out experiments using Trp 1798 as a reporter of its environment.…”

Section: Thermodynamic Cycle Analysis Is Consistent With Predictedmentioning

confidence: 99%

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A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Glaaser

Bankston

Liu³

et al. 2006

Journal of Biological Chemistry

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Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart Na V 1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the Na V 1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na ؉ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na ϩ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2-4). Clinical consequences of inherited mutations that disrupt Na ϩ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins.In the heart, Na ϩ channels (Na V 1.5) 4 primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for Na V 1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5), and in most cases, these inherited mutations disrupt channel inactivation.Fast inactivation of Na ϩ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization...

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Section: Thermodynamic Cycle Analysis Is Consistent With Predictedsupporting

confidence: 84%

Section: Thermodynamic Cycle Analysis Is Consistent With Predictedmentioning

confidence: 99%

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Glaaser

Bankston

Liu³

et al. 2006

Journal of Biological Chemistry

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“…However, a marked variation between various polyclonal IgG preparations was observed regarding the extent of acrylamide quenching. Acrylamide has been reported to have access to relatively buried tryptophans (Eftink and Ghiron, 1977 ;Have1 et al, 1988), although truly buried tryptophan residues are not measurably quenched by acrylamide and other polar quenchers (Eftink and Ghiron, 1976;Calhoun et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

“…An increase in Ksv suggests a higher internal flexibility of the amino acid residues. The higher the dynamic fluctuation of the protein matrix, the higher the probability that transient channels open and therefore allow a quencher to be entrapped (Eftink and Ghiron, 1977;Calhoun et a]., 1983;Eftink and Hagaman, 1986;Somogyi et al, 1986). Furthermore, the increase in f a for iodide when lowering the pH means that more tryptophan residues become accessible to iodide.…”

Section: Discussionmentioning

confidence: 99%

Non‐random conformation of a mouse IgG_2a monoclonal antibody at low pH

Jiskoot¹,

Bloemendal²,

Haeringen³

et al. 1991

European Journal of Biochemistry

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The pH dependence of the conformation of a mouse IgG2,,, monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 ps) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 ps; a further increase to 0.30 ps was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.Monoclonal antibodies (mAbs) are immunoglobulins, usually of the IgG class, consisting of two identical heavy and two identical light chains which are covalently linked by disulphide bonds and folded into compact globular regions. In spite of a nearly unrestricted variety of mAbs that can be elicited against almost any molecule, their overall structure is very similar and consists of several homologous domains of highly conserved amino acid sequences; six short regions (about ten amino acid residues each) of highly variable amino acid composition form the antigen-binding site. The immunoglobulin domains are made up of P-pleated sheets and contain little, if any, %-helical structures. Whereas the individual IgG domains may be regarded as rigid structures, segmental flexibility, particularly in the hinge region, has been documented and is probably of functional importance (Huber, 1987). Comprehensive reviews about structure and function of immunoglobulins have been given by Jeske and Capra (1984) and by Edmundson and Ely (1986). mAbs belong to the new generation of biotechnological products that are becoming increasingly important in pharmaceutical sciences (Robinson, 1988;Sherwood, 1988 An important aspect of pharmaceutical mAb preparations is their physico-chemical stability upon storage (Manning et al., 1989). To be able to guarantee the physical and chemical integrity of complex proteins such as mAbs, the availability and application of an arsenal of relevant analytical techniques is a prerequisite. In a previous article (Jiskoot et al., 1990) it was demonstr...

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“…NATA was used as a control material for fluorescence experiments, as it is an indole derivative that closely mimics a tryptophan residue involved in peptide bonds (21,30,31). NMR Spectroscopy-Samples for NMR measurements contained 3 mM peptide in a 300 mM SDS-d 25 solution containing 7% D 2 O at pH 4.0.…”

Section: Methodsmentioning

confidence: 99%

Systematic Peptide Engineering and Structural Characterization to Search for the Shortest Antimicrobial Peptide Analogue of Gaegurin 5

Won

Jung

Kim

et al. 2004

Journal of Biological Chemistry

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As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.Most organisms produce membrane-active peptides that exhibit antibiotic, fungicidal, hemolytic, virucidal, and tumoricidal activities (1, 2). It is becoming clear through many studies that antimicrobial peptides are an important component of the innate defenses of all species of life (3-8). Antimicrobial peptides function by interacting with the cell membrane (1-6). In particular, cationic, linear, helical peptides, which are the most well characterized group of antimicrobial peptides, function by the "barrel-stave" and/or "carpet-like" mechanism, leading to bacterial membrane permeation (1, 5, 9 -13). In membrane environments, the peptides generally adopt an amphipathic helical structure, positioning the hydrophobic residues on one side and the hydrophilic residues on the other side of the helical axis, whereas they assume a random coil conformation in aqueous solutions.Recently, antimicrobial peptides have become a potential source of new antibiotics to combat the increasing emergence of drug-resistant bacteria (4, 5). Several antimicrobial peptides such as magainin, a 23-residue antimicrobial peptide, have been successful in pharmaceutical and commercial development (5). Particularly, from the first discovery of bombinins in Bombina variegata (7), the skin of anurans (frogs and toads) has proven to be a rich source of peptides with ...

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Exposure of tryptophanyl residues and protein dynamics

Cited by 297 publications

References 23 publications

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Non‐random conformation of a mouse IgG_2a monoclonal antibody at low pH

Systematic Peptide Engineering and Structural Characterization to Search for the Shortest Antimicrobial Peptide Analogue of Gaegurin 5

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Exposure of tryptophanyl residues and protein dynamics

Cited by 297 publications

References 23 publications

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Non‐random conformation of a mouse IgG2a monoclonal antibody at low pH

Systematic Peptide Engineering and Structural Characterization to Search for the Shortest Antimicrobial Peptide Analogue of Gaegurin 5

Contact Info

Product

Resources

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Non‐random conformation of a mouse IgG_2a monoclonal antibody at low pH