The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature T m , of thermally induced denaturation of STV from 75°C in unliganded protein to 112°C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a K a ϳ10 15 reproduce the behavior observed, but the estimated values of T m are 15-20°C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.The biological function of many proteins is triggered and modulated by the binding of ligands. For this reason, an understanding of the mechanism of protein-ligand interactions is essential for a detailed knowledge of protein function at the molecular level. Ligand binding, in most cases, involves the formation of noncovalent bonds at specific interacting surfaces between the protein and the ligand. The binding of a ligand can be accompanied by conformational changes at the protein site that sometimes are propagated throughout the entire protein.It is desirable to have a way to monitor these structural changes to understand any new properties acquired by the complex. The high affinity of the biotin-streptavidin binding not only offers useful bioanalytical advantages (1), but it also makes this system an attractive model for studying proteinligand interactions (2-5). The biotin⅐STV 1 association constant of about 10 15 is the highest known in biochemistry. In the present work we explore protein thermostability by heating STV in the absence of ligand or under conditions of partial or full ligand saturation. A dramatic increase in the T m of protein denaturation, from 75°C in absence of biotin to 112°C at full ligand saturation, was revealed using differential scanning calorimetry (DSC). An analysis of the cooperativity of the denaturation was made on the basis of a reversible nontwo-state model of protein unfolding to gain understanding of the system that unfolds differently if biotin is present.Conformational changes were characterized by FT-IR and fluorescence spectroscopy. The large changes in the...

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.

The underlying noise in the infrared spectra of proteins may introduce artifacts in the quantitation of proteins by curve‐fitting of the amide I band. Smoothing methods are able to reduce the noise but can introduce alterations in band shape that affect the information contained in the spectrum. Three methods to remove noise—Savitzky‐Golay, Fourier filtering, and maximum entropy—have been used to ascertain their influence on the quantitative information when applied to protein bands. Use of artificial curves shows that whereas Savitzky‐Golay and Fourier smoothing are able to reduce the noise, they distort the band shape. Maximum entropy is more efficient in reducing the noise in artificial curves with added noise, and provided a narrowest bandwidth below 12 cm−1, no band‐shape distortion is obtained. Using the smoothing in natural spectra, the presence of spurious bands in the initial parameters coming from artifacts introduced by deconvolution or derivation is reduced. Moreover, the dispersion in the percentage area values in a series of similar spectra is also decreased below 2%, a value that discriminates the effect of ligand binding to proteins. The maximum entropy method is proposed as a tool to improve the quantification of protein structure by infrared spectroscopy. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 469–475, 1997

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Sarcoplasmic reticulum Ca2+-ATF'ase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as a-helix, P-sheet, etc. . . . , two unusual bands, one at 1,645 cm" in H 2 0 buffer and the other at 1,625 cm" in D 2 0 buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm" is tentatively assigned to a-helical segments located in the cytoplasmic domain and coupled to /?-sheet structure, whereas the band at 1,625 cm" arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% a-helical segments in the transmembrane plus stalk domain; 20% a-helix and 22% @sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca*+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm" band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm" band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37"C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.Keywords: Ca2+-ATPase; infrared spectroscopy; protein structure; proteolysis; sarcoplasmic reticulum; thermal analysis Knowledge of the structure-function relationship is essential in understanding the molecular mechanisms underlying the membranecontrolled biological processes. The X-ray three-dimensional structure of membrane proteins is still not well known, except for a few cases. Therefore, other lower resolution spectroscopic methods have to be used to gain insight of the structure and function of membrane proteins. Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein that pumps calcium out of the cytoplasm during striated muscle relaxation (Martonosi, 1996). This ATPase is part of a family of P-type ion pumps that includes several cation-activated ATPases having in common ten predicted transmembrane helical segments (Stokes et al., 1994).The structure of sarcoplasmic reticulum Ca2+-ATPase has been predicted from the amino acid sequence (MacLennan et al., 1985) and from electron microscopy observations (Toyoshima et al., 1993). The protein appears to consist of an extensive beak-shaped cytoplasmic domain containing interconnected a-helical and P-strand segments, a stalk connecting the beak with the membrane, and the ten transmembrane segments characteristic of P-type ion pumps (Stokes et al., 1994). The cytoplasmic domain contains the active sites of ATP hydrolysis and phosphorylation while the Ca2+ channel is expected to be associated to the transmembrane domain. Some Ca2+-...