Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

Mark C. Williams

Kiran Pant

Ioulia Rouzina

Richard L. Karpel

Single molecule force spectroscopy studies of DNA denaturation by T4 gene 32 protein

Insulin&#8211;like growth factor&#8211;I (IGF&#8211;I) is a central mediator of cell growth, differentiation and metabolism. Structural characterization of the protein has been hampered by a combination of internal dynamics and self&#8211;association that prevent crystallization and produce broad NMR resonances. To better characterize the functions of IGF&#8211;I, we have used phage display to identify peptides that antagonize the binding of IGF&#8211;I to its plasma binding proteins (IGFBPs) and cell&#8211;surface receptor (IGF&#8211;R). Interestingly, binding of peptide improves dramatically the quality of the NMR resonances of IGF&#8211;I, and enables the use of triple&#8211;resonance NMR methods to characterize the complexes. One such peptide, designated IGF&#8211;F1&#8211;1, has been studied in detail. In the complex, the peptide retains the same loop&#8211;helix motif seen in the free state whilst IGF&#8211;I contains three helices, as has been seen previously in low&#8211;resolution structures in the absence of ligand. The peptide binds at a hydrophobic patch between helix 1 and 3, a site identified previously by mutagenesis as a contact site for IGFBP1. Thus, antagonism of IGFBP1 binding exhibited by the peptide occurs by a simple steric occlusion mechanism. Antagonism of IGF&#8211;R binding may also be explained by a similar mechanism if receptor binding occurs by a two&#8211;site process, as has been postulated for insulin binding to its receptor. Comparisons with crystallographic structures determined for IGF&#8211;I in other complexes suggest that the region around helix 1 of IGF&#8211;I is conformationally conserved whereas the region around helix 3 adopts several different ligand&#8211;induced conformations. The ligand&#8211;induced structural variability of helix 3 appears to be a common feature across the insulin super&#8211;family. In the case of IGF&#8211;I, exchange between such conformations may be the source of the dynamic nature of free IGF&#8211;I, and likely has functional significance for the ability of IGF&#8211;I to recognize two signaling receptors and six binding proteins with high affinity.

Nicholas J. Skelton

Michelle L. Schaffer

Kurt Deshayes

Tamas Blandl

Steven Runyon

Gerald Nakamura

Sachdev S. Sidhu

Phage display-derived ligands provide structural insight into insulin-like growth factor I function

Lipoxygenases (Loxs) form a homologous family of non-heme, non-sulfur iron containing lipid-peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed “mini-Lox”, which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi-Agrò, Biochemistry40 (2001), 6819–6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition. Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency.

Mauro Maccarrone

Almerinda Di Venere

Guus van Zadelhoff

Giampiero Mei

Gerrit Veldink

Nicola Rosato

Alessandro Finazzi-Agrò

Further structural and functional properties of mini‒lipoxygenase, an active fragment of soybean lipoxygenase-1

This work introduces a new approach connecting vibrational spectroscopy with homology and energetic molecular modeling of proteins. Combination of both methods can compensate their disadvantages and result in realistic three-dimensional protein models. The approach is most powerful for membrane proteins or glycoproteins with high carbohydrate content where X-ray or NMR analysis is not always successful. Nevertheless, it can also serve as a tool of preliminary analysis of any protein with unknown structure. Power of the approach is demonstrated on human &#945;1-acid glycoprotein. Its predicted structure published in [V. Kopeck&#253; Jr. et al., Biochem. Biophys. Res. Commun. 300 (2003), 41&#8211;46] is discussed in detail with respect to the approach and its general employment.

Vladim&#237;r Kopeck&#253;

R&#252;diger Ettrich

Kate&#x159;ina Hofbauerov&#225;

Vladim&#237;r Baumruk

Vibrational spectroscopy and computer modeling of proteins: solving structure of &#945;1-acid glycoprotein

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IP state) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IP state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IP state of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IP state is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.

Yuji O. Kamatari

Christopher M. Dobson

Takashi Konno

Structural dissection of alkaline-denatured pepsin

The effect of salts, detergents and chaotropic agents on mass spectrometric analysis are relatively well understood, mainly due to their actions decreasing the performance of ESI interface in mass spectrometric analysis. However, there are few studies in the literature characterizing the effect of protein stabilization by glycerol, followed in some circumstances by the suppression of protein signal when ESI interface is used. The aim of the present research was to investigate in details the mass spectrometric behavior of some proteins in presence of high levels of glycerol during ESI–MS analysis. Thus, horse heart myoglobin and chicken ovalbumin were used as standard proteins. It was demonstrated that the presence of 1% (v/v) glycerol suppressed the signal of these proteins during the ESI–MS analysis, even when the sample nozzle potential was scanned from 28 to 80 V. However, when the glycerol concentration was decreased to 0.5% (v/v) and the sample cone voltage adjusted to 50 V, a perfect envelope of peaks was observed, allowing the spectrum deconvolution and the molecular mass determination with mass accuracy lower than 0.01% in each situation. A molecular explanation for this suppressive effect and for the analytical overcoming of this difficult is proposed.

M. A. Mendes

B. M. Souza

M. R. Marques

M. S. Palma

The effect of glycerol on signal supression during electrospray ionization analysis of proteins

The azaspiracids, AZA1, AZA2 and AZA3, are the predominant shellfish toxins responsible for the human toxic syndrome, azaspiracid poisoning. Collision induced dissociation (CID) mass spectra were generated for azaspiracids using nano-electrospray ionisation (ESI) with a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer in positive mode. Six main backbone fragmentations of the polyether skeleton of azaspiracids were observed as well as multiple neutral losses of water molecules from the parent and product ions. The characteristic charge-remote fragmentation of the carbon skeleton of azaspiracids produced nitrogenous ions. The three azaspiracids differ from one another by 14 Da due to methylation in the A- and E-rings. Three fragmentation pathways, involving cleavage of the E-ring, C27&#8211;C28 and G-ring, gave ions that were common to all azaspiracids. Another three fragmentations involving the A-ring, C-ring and C19&#8211;C20, were useful for distinguishing between azaspiracid analogues. Multiple tandem ion‒trap mass spectrometry (MSn) was used to confirm the fragmentation pathways.

Brett Hamilton

M&#243;nica D&#237;az Sierra

Mary Lehane

Ambrose Furey

Kevin J. James

The fragmentation pathways of azaspiracids elucidated using positive nanospray hybrid quadrupole time-of-flight (QqTOF) mass spectrometry

The potential of a sustained release formulation for paracetamol produced by melt pelletisation was investigated. After the production of the pellets, based on the combination of stearic acid as a melting binder and anhydrous lactose as a filler, the 3000–2000 μm size fraction was selected in the light of the promising in vitro dissolution results for further characterisations, including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), specific surface area and true density determination. Hence the release mechanism was analysed with the help of an appropriate mathematical model. The mathematical model was built on the hypotheses that drug diffusion and solid drug dissolution in the release environment are the key phenomena affecting drug release kinetics. Bioavailability of the developed formulation was evaluated in an in vivo study in eight subjects.

Mario Grassi

Dario Voinovich

Iztok Grabnar

Erica Franceschinis

Beatrice Perissutti

Jelena Filipovic-Grcic

Preparation and in vitro/in vivo characterisation of a melt pelletised paracetamol/stearic acid sustained release delivery system

The quantification of boron neutron capture therapy (BNCT) 10B‒carrier, L‒p‒boronophenylalanine‒fructose complex (BPA–F) was studied with phantoms using 1H magnetic resonance spectroscopy sequences PRESS and STEAM at 1.5 and 3.0 T. The results show that typical attainable short echo times of clinical MRS sequences combined with long repetition time result in clinically acceptable quantification accuracy. However, the concentration ratios, which are essential for the treatment planning, can still be reliably measured by using small repetition times. Detection limits of BPA in aqueous phantoms at 1.5 and 3.0 T were evaluated using clinically acceptable measurement time of ~10 min, two typical voxel sizes (153 and 203 mm3) and PRESS and STEAM sequences. The detection limits of BPA in phantom conditions were 0.7 (3.0 T) and 1.4 mM (1.5 T) for PRESS sequence with 203 mm3 voxel.

M. Timonen

A. Kangasmäki

S. Savolainen

S. Heikkinen

1H MRS phantom studies of BNCT 10B‒carrier, BPA–F using STEAM and PRESS MRS sequences: Detection limit and quantification

Annelids are capable of defending themselves from pathogens and of recognizing degenerated self tissue. These reactions require specialized immune mechanisms that are effected by proteins and cellular reactions. Hemolytic proteins are the most striking humoral defense molecules in the earthworm Eisenia fetida. Beside their hemolytic activity these proteins possess agglutinating, antibacterial, cytotoxic, and clotting properties. Hemolytic proteins both from coelomocytes (CL39,41) and coelomic fluid (H1−3) of wildtype E. fetida were isolated and assigned to fetidin and lysenin using mass spectrometry and bioinformatic tools. Glycosylation was found for H1. In silico analyses of the hemolysins revealed two hemolysin isoforms and consensus sites for N-glycosylation and peroxidases proximal heme-binding ligand.

Simone König

Frank Wagner

Ellen Kauschke

Ines Eue

Sequence analysis of earthworm hemolysins

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Journal of Spectroscopy publishes research into the theory and application of spectroscopy across all disciplines, including biology, chemistry, engineering, earth sciences, medicine, materials science, physics, and space science.

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Chief Editor&nbsp;Dr Daniel Cozzolino&nbsp;is based at the University of Queensland, Australia. His research focuses on the developments of chemometric and spectroscopic methods for use in agriculture and food applications.

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