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+-...

The secondary structure of wild-type Paracoccus denitrificans cytochrome c oxidase obtained by decomposition of the infrared amide I band contains 44% alpha-helix, 18% beta-sheet, 14% beta-turns, 18% loops, and 6% nonordered segments. The mutant lacking subunit III presents a small but significant increase (from 18% to 24%) in the percentage of loops and slight differences in the other components. Using band/area ratios and tyrosine side chain absorption as an inner standard, it is shown that in the absence of subunit III the structure of subunits I and II is altered although no changes in their alpha-helix or beta-sheet content are observed. In the bacterial oxidase, thermal infrared studies show a complex denaturation pattern characterized by the presence of a partially denatured intermediate state. Of the seven predicted subunit III alpha-helices, only four are resistant toward the thermal challenge and behave as expected for typical transmembrane helices. The observation that the absence of subunit III influences the conformation of loop regions in the two other subunits suggests that part of the interaction surface between subunit III and the catalytic subunits might be located outside the lipid bilayer.

Mixtures of di-(perdeuteropalmitoy1)-sn-glycero-3 choline ( [ZH6,]Pam,GroPCho) with palmitoylcarnitine or palmitoyl-CoA in aqueous suspension have been examined by Fourier-transform infrared spectroscopy. The C-H (or C-D) region of the spectrum shows that an order-disorder transition exists in pure aqueous palmitoylcarnitine at 45 "C ; palmitoylcarnitine mixes with [ZH6,]Pam,GroPCho without perturbing the gel-fluid transition of the phospholipid even at [2H,,]PamZGr~PCho/palmitoylcarnitine 1 : 2 molar ratios ; and palmitoyl-CoA, however, at similar proportions, smears out the [ZH,]Pam,GroPCho transition as detected from C-D stretching vibrations. Relevant data from the carbonyl region include ; the high-frequency (non-hydrogen bound) carbonyl subpopulation, but not the low frequency one, detects the gel-to-fluid transition of the phospholipid; the carbonyl region detects the thermotropic transition over a wider temperature range than the methylene stretching region, i.e. detects changes starting well below and ending several degrees above the methylene transition temperature, and a significant interaction may occur between some coenzyme A group and the carbonyl groups of the phospholipid. The latter interaction may contribute to explain the coenzyme Ncamitine exchange during mitochondrial fatty acid import.Keywords. Carnitine, fatty acyl derivatives ; coenzyme A, fatty acyl derivatives ; infrared spectroscopy ; phase transitions.In eukaryotes, fatty acids are degraded in the mitochondrial matrix in the form of coenzyme A derivatives. They exist in the same form in the cytosol, but their import across the inner mitochondrial membrane involves their transient and reversible conversion into fatty acylcarnitines [ 11. The physico-chemical basis for such coenzyme exchange has not been clearly established. Thus, a number of studies have been recently carried out in our laboratory in which the effects of palmitoyl-CoA and palmitoylcarnitine, representing, respectively, the fatty acylCoAs and fatty acylcarnitines, on various model membranes have been comparatively assessed.Langmuir trough measurements of the penetration of palmitoyl-CoA (PamCoA) and palmitoylcarnitine into egg phosphatidylcholine (PtdCho) monolayers [2] indicated that the coenzyme A derivative mixed ideally with phospholipids, while palmitoylcarnitine bound phosphatidylcholine in a cooperative way. A more recent comparative study of the interactions of palmitoylCoA and palmitoylcarnitine with egg PtdCho liposomes [3] shows that the latter acts very much as a surfactant, achieving complete solubilization of the lipid bilayers at surfactanthpid molar ratios above 2. Pam-CoA also binds the bilayer with high affinity but no further membrane effect ensues. In view of these data, a more detailed study of the molecular interactions of PtdCho bilayers with both kinds of fatty acyl derivatives was undertaken, using infrared spectroscopy. This technique has been repeatedly shown as appropriate in the study of membraneamphiphile interactions [4, 51. The results...

The TrwC protein is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. The TrwC-N275 fragment comprises the 275-amino-acid Nterminal domain of the protein that contains the DNA cleavage and strand transfer activities (the relaxase domain). It can be easily purified by keeping a cell lysate at 90°C for 10 min. Infrared spectroscopy shows that this domain has a predominantly ␣/␤ structure with some amount of unordered structure. Fast heating and cooling does not change the secondary structure, whereas slow heating produces two bands in the infrared spectrum characteristic of protein aggregation. The denaturation temperature is increased in the protein after the fast-heating thermal shock. Two-dimensional infrared correlation spectroscopy shows that thermal unfolding is a very cooperative two-state process without any appreciable steps prior to aggregation. After aggregation, the ␣-helix percentage is not altered and ␣-helix signal does not show in the correlation maps, meaning that the helices are not affected by heating. The results indicate that the domain has an ␣-helix core resistant to temperature and responsible for folding after fast heating and an outer layer of ␤-sheet and unordered structure that aggregates under slow heating. The combination of a compact core and a flexible outer layer could be related to the structural requirements of DNA-protein binding.Bacterial conjugation is the result of a two-step process, DNA processing and DNA transport. Each step is carried out by a specific set of proteins encoded by the tra genes of a given conjugative plasmid (16). Conjugation begins with the cleavage of the donor supercoiled DNA by a specific relaxase and the formation of a nucleoprotein complex, the relaxosome, which contacts the transport site. A multiprotein DNA transport system effects the transfer process of the cleaved DNA strand to the recipient cell. The relaxase religates the transferred DNA strand upon transfer, and finally, host proteins replicate both single strands in the donor and recipient bacteria to regenerate the double-stranded conjugative plasmid.Relaxases are classified into five families according to the DNA sequences around the nic sites and their amino acid sequences (9). The F family relaxase TrwC, from the IncW plasmid R388, is a dimeric protein of 996 amino acids in which the N-terminal domain has a DNA relaxase activity and the C-terminal domain is a DNA helicase (18). In addition to relaxase and helicase activities, the TrwC protein promotes site-specific recombination between oriT sequences in vivo. In addition, it can cleave a supercoiled plasmid DNA containing oriT in vitro in the absence of accessory proteins (17).Infrared (IR) spectroscopy has become a widely used tool in the study of protein structure. In principle, a structure as large as a protein would give rise to an enormous number of overlapping vibrational modes, obscuring the information that could be obtained in practice, but because o...