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Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.

Oritavancin is a semi-synthetic glycopeptide antibiotic which is structurally related to vancomycin. When oritavancin bisphosphate is dried in vacuo with heat, a new compound forms. This new compound is stable only in the solid state and reverts to oritavancin in solution. Highly enriched samples of this compound were obtained by preparative HPLC and the structure of this compound was elucidated by using one and two-dimensional ( 1 H and 13 C) NMR spectroscopy in conjunction with computer-assisted molecular modeling. It has been determined that oritavancin adopts a conformation similar to that of vancomycin in solution, while the new compound is the unnatural R -AB-biaryl atropisomer of oritavancin. This is the first observation and isolation of an AB-biaryl atropisomer in an intact member of the vancomycin family of glycopeptide antibiotics.

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.

Inhibition of urokinase activity represents a promising target for antimetastatic therapy for several types of tumor. The present study sets out to investigate the potential of Raman spectroscopy for defining the molecular details of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstrated that high quality Raman spectra from a series of five inhibitors bound individually to the active site of human urokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope. After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional group on a naphthalene ring was added, and the spectrum of the crystal−inhibitor complex was obtained. The resulting difference Raman spectrum contained only vibrational modes due to bound inhibitor, originating from the protonated group, i.e., the amidinium moiety, as well as naphthalene ring modes and features from other functionalities that made up each inh...

The coupling of apoptosis (programmed cell death) to the cell division cycle is essential for homeostasis and genomic integrity. Here, we report the crystal structure of survivin, an inhibitor of apoptosis, which has been implicated in both control of cell death and regulation of cell division. In addition to a conserved N-terminal Zn finger baculovirus IAP repeat, survivin forms a dimer through a symmetric interaction with an intermolecularly bound Zn atom located along the molecular dyad axis. The interaction of the dimer-related C-terminal α helices forms an extended surface of ∼70 A in length. Mutagenesis analysis revealed that survivin dimerization and an extended negatively charged surface surrounding Asp-71 are required to counteract apoptosis and preserve ploidy. These findings may provide a structural basis for a dual role of survivin in inhibition of apoptosis and regulation of cell division.

Determination of the oligomeric state of integral membrane proteins in detergent solutions is a challenging task because the amount of detergent associated with the protein is typically unknown and unpredictable. Methods that estimate the molecular weight of proteins from their hydrodynamic properties in solution are not suitable for detergent-solubilized membrane proteins. However, size-exclusion chromatography (SEC) performed in combination with analyses of static light scattering (SLS), ultraviolet absorbance (UV), and refractive index (RI) provides a universal method for determination of the molar masses of biopolymers and protein-detergent complexes. The light scattered by a protein is directly proportional to its molecular mass, irrespective of shape, and any additional contributions due to bound detergent molecules can be quantitatively accounted for by the additional combined analysis of ultraviolet absorbance and refractive index information. The primary intention of this unit is to describe how to apply the combination of high-performance liquid chromatography SEC and SLS-UV-RI to evaluate molecular mass and the physicochemical heterogeneity of purified membrane protein-detergent complexes.

The equilibrium folding behavior of recombinant human FK-binding protein, a peptidyl-prolyl cis-trans-isomerase, was examined by urea-induced denaturation using probes of protein structure including intrinsic tryptophan fluorescence, second-derivative UV absorbance, CD, and NMR. All optical probes of protein structure indicate that FKBP is capable of folding reversibly. The second-derivative UV absorbance and CD probes of the structure exhibited urea denaturation transitions at approximately 4.3 M urea. The fluorescence of the single protein tryptophan is quenched in the folded state. During the unfolding-folding transition, the unquenching of tryptophan fluorescence occurs at a slightly lower urea concentration (3.9 M urea) than the changes observed for the other optical probes of folding. These probes of structure demonstrate little dependence on protein concentration in the range of 0.2- approximately 3 mg/mL across the urea-induced denaturation transition. The reversibility of the unfolding-folding transition was confirmed from two-dimensional 15N/1H heteronuclear single-quantum coherence (HSQC) spectra of [U-15N]FKBP. In addition, the native-denatured transitions for 57 individual amino acids were determined from an analysis of these spectra acquired at different urea concentrations. Analysis of the transitions for all clearly observable HSQC cross peaks for residues distributed throughout the protein and comparison to the optical folding transitions, indicate that FKBP global folding is consistent with a two-state process. Although direct measurement of FKBP catalytic activity in urea was complex, enzyme activity was observed up to the beginning of the FKBP urea-denaturation transition.(ABSTRACT TRUNCATED AT 250 WORDS)

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide 1 is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

Time-resolved phosphorescence anisotropy has been used to study the rotational diffusion of eosin-labeled human erythrocyte band 3 in the presence of an enzyme bound at its cytoplasmic pole. With increasing amounts of G3PD (glyceraldehyde-3-phosphate dehydrogenase) added to ghosts, the infinite time anisotropy (r infinity) increases, and at saturating concentrations, very little decay of the anisotropy r(t) occurs at all. These phenomena are reversed by elution of the enzyme with 150 mM NaCl. Prior proteolytic removal of the N-terminal 41-kDa cytoplasmic fragment of band 3 also disenables the G3PD effect. When ghosts are stripped of their residually bound G3PD, a small reduction in the fraction of immobile band 3 is observed. Finally, titration of band 3 sites with aldolase shows effects on the r(t) qualitatively similar to those observed with G3PD. On the basis of our interpretation of the heterogenous anisotropy decay of eosin-labeled band 3 [Matayoshi, E. D., & Jovin, T. M. (1991) Biochemistry (preceding paper in this issue)], we conclude that the binding of G3PD and aldolase results in the immobilization of band 3 oligomers.

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira(TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel(TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.© (2008) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The design and synthesis of new fluorogenic substrate probes for HIV protease based on resonance energy transfer arc described. These substrates permit sensitive, continuous measurement of HIV protease activity.

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the “Trp-Trp-Ile” binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

The human immunodeficiency virus type 1 (HIV-1) gp41 plays an important role in mediating the fusion of HIV with host cells. During the fusion process, three N-terminal helices and three C-terminal helices pack in an anti-parallel direction to form a six-helix bundle. X-ray crystallographic analysis of the gp41 core demonstrated that within each coiled-coil interface, there is a deep and large pocket, formed by a cluster of residues in the N-helix coiled-coil. In this report, we systematically analyzed the role of seven conserved residues that are either lining or packing this pocket on the infectivity and interhelical interaction using novel approaches. Our results show that residues L568, V570, W571, and K574 of the N-helix that are lining the side chain and right wall of the pocket are important for establishing a productive infection. Mutations V570A and W571A completely abolished replication, while replication of the L568A and K574A mutants was significantly attenuated relative to wild type. Similarly, residues W628, W631, and I635 of the C-helix that insert into the pocket are essential for infectivity. The impaired infectivity of these seven mutants is in part attributed to the loss in binding affinity of the interhelical interaction. Molecular modeling of the crystal structure of the coiled-coil further shows that alanine substitution of those residues disrupts the hydrophobic interaction between the N- and C-helix. These results suggest that the conserved residues in the coiled-coil domain play a key role in HIV infection and this coiled-coil pocket is a good target for development of inhibitors against HIV. In addition, our data indicate that the novel fluorescence polarization assay described in this study could be valuable in screening for inhibitors that block the interhelical interaction and HIV entry.

Fluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques. This paper reviews the general protocol for application of FCS to biomolecular compound-binding assays and it focuses on methods for the reduction of experimental photon count data to obtain the normalized autocorrelation function (ACF), on theoretical models of the ACF, and on statistical and systematic errors in the experimental ACF. Results from a detailed Monte Carlo simulation of FCS, which are useful for testing theoretical models and validating short-duration assay capabilities, are discussed. An illustrative example is presented on the use of FCS to assay binding of Alexa-488-labeled Bak peptide with Bcl-x(L), which is an intracellular protein that acts to protect against programmed cell death.

Fluorescence Correlation Spectroscopy (FCS) may be used to assay the binding of drug-like ligands to signaling proteins and other bio-particles. For High Throughput Screening (HTS), a competitive format is typically used in which the binding of an unlabeled compound results in displacement of a fluorescently labeled ligand. Unweighted curve-fitting of the normalized autocorrelation function (ACF) to a two-diffusion-component model can resolve the fractions of free and bound ligand if the diffusion rates differ sufficiently and if the experimentally estimated ACF has adequate precision. However, for HTS (and also for intracellular FCS studies) it is desirable to minimize the experimental data collection time. In this case, the precision of the ACF is limited and it becomes important to account for the statistical features of the ACF estimate when designing an assay. The errors at different points in the estimated ACF are correlated and hence least-squares fitting methods are not statistically rigorous. We compare different methods for estimating and curvefitting the ACF from the raw data of short duration FCS measurements. The methods are applied to data from experiments to assay binding of Alexa-488-labeled Bak peptide with Bcl-xL, which is an intracellular protein that acts to protect against programmed cell death. We present results from a detailed Monte Carlo simulation of the experiment, which is useful for validating short-duration assay capabilities. We also discuss the measurement of changes in steady state fluorescence anisotropy due to restricted rotational diffusion upon binding, which provides a complementary assay. 1. USE OF FLUORESCENCE POLARIZATION AND FLUORESCENCE CORRELATION SPECTROSCOPY FOR DRUG-BINDING ASSAYS IN HIGH-THROUGHPUT SCREENING The initial task in high-throughput pharmaceutical drug screening is to sort through hundreds of thousands of drug-like library compounds to find those that bind or interact with a target protein or bio-molecule, which has been identified from prior molecular-biology knowledge [1]. Miniaturization of sample volumes to conserve reagents and homogeneous solution assay are generally preferable in the initial screening measurements. The overall time for high throughput screening (HTS) may be reduced by multiplexing the initial measurements. For example, each sample may contain many different compounds, with each compound occurring in two or more different samples, and matching of the binding assay hits may be used to identify the particular compound responsible for each hit. Further measurements and research can then be performed on the identified compounds. Figure 1 illustrates typical data from a HTS run. An experimental measure is made of the degree of binding to the target that occurs for any of the compounds within each sample. Systematic errors in the binding measure are oftentimes present. These may arise from many causes, including precipitation of solution components, uncertainties in the concentrations of solution components, unwanted interactions between test compounds, and unaccounted sources of signal such as autofluorescence [1]. Such errors account for apparent binding results that are less than 0%, or greater than 100%, as seen in Figure 1. Nevertheless, it is still possible to identify many compounds which consistently yield apparent binding, and which warrant further assays. However, these hits are usually only expected to lead synthetic chemists toward new and better compounds. The success of the drug discovery/design process depends on further medicinal chemistry and structural biology.

In this work we characterized seven dyes that are one-photon excited and fluoresce in the far-red region of the visible spectrum, five of which became available only recently, as to their suitability as labels on the red emission channel in a two-color FSC method. Spectroscopic properties and binding to albumin were studied. In addition, when one of these was used as the label in a first-pass high-throughput screen with single-color fluorescence polarization detection, its foreseen advantage of avoiding the excitation of interfering background autofluorescence from compounds in libraries was deemed valuable after a comparison of two complete high-throughput screens of a chemical compound library at Abbott.

The three-dimensional structure of the anti-apoptotic protein Bcl-xL complexed to a 25-residue peptide from the death promoting region of Bad was determined using NMR spectroscopy. Although the overall structure is similar to Bcl-xL bound to a 16-residue peptide from the Bak protein (Sattler et al., 1997), the Bad peptide forms additional interactions with Bcl-xL. However, based upon site-directed mutagenesis experiments, these additional contacts do not account for the increased affinity of the Bad 25-mer for Bcl-xL compared to the Bad 16-mer. Rather, the increased helix propensity of the Bad 25-mer is primarily responsible for its greater affinity for Bcl-xL. Based on this observation, a pair of 16-residue peptides were designed and synthesized that were predicted to have a high helix propensity while maintaining the interactions important for complexation with Bcl-xL. Both peptides showed an increase in helix propensity compared to the wild-type and exhibited an enhanced affinity for Bcl-xL.

We will discuss several examples from our laboratory of the application of fluorescence correlation spectroscopy (FCS) to studies on protein-ligand interactions in free solution. To demonstrate the usefulness of FCS in such investigations we present situations which span both ligand size (from small molecules of molecular weight MW ≈ lkDa, to large protein ligands) and affinity (picomolar to micromolar).Furthermore, since FCS technology within the biological research community is available in only a small number of labs, we compare the results obtained by FCS with those obtained on the same samples by fluorescence polarization (FP). Such a comparison is useful because FP is a general and well-established optical biophysical tool for homogeneously monitoring molecular associations. While there clearly is overlap in the types of problems amenable to FCS and FP, the information they provide is complementary, and we believe it will be desirable to use them in tandem whenever feasible. We begin by briefly reviewing and comparing FCS and FP.

The possibility and potential benefits of using an extreme red fluorescent dye such as Cy5.5TM to label drug discovery target proteins was studied experimentally. Cy5.5 labeled BSA, GFP, and CRP were used as examples of protein-dye conjugates whose binding to corresponding antibody was detected by changes in either rotational or transnational diffusion properties, that is, by either fluorescence polarization or confocal fluorescence correlation spectroscopy (FCS). In addition, FCS was used to quantitate excitation of Cy5.5 to its triplet state. Fluorescence polarization and lifetime were measured as a function of excitation wavelength or glycerol concentration and solvent viscosity.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The rotational diffusion of eosin-labeled 3 in human erythrocyte cells and hemoglobin-free ghosts at 37 degrees C has been studied in detail by polarized delayed luminescence. The time-resolved anisotropy with both cells and freshly prepared ghosts is similar, decaying with well-resolved rotational correlation times of 0.03, 0.2, and greater than or equal to 1 ms. Mild proteolytic removal of the water-soluble 41-kDa cytoplasmic domain of band 3 in ghosts results in a drastic increase in the fractional contributions of the two fastest depolarizing components. Our results, taken together with other data in the literature, imply that several classes of band 3 that differ greatly in mobility exist in ghosts and intact cells. The mobility of one class is hindered due to complexation with other membrane or cytoplasmic proteins mediated via the 41-kDa cytoplasmic domain. However, another class of band 3 molecules exists as homo-or heterooligomeric complexes larger than a dimer that are stabilized by hydrophobic interactions involving the intramembranal domain. Finally, the presence of the (previously undetected) 0.03-ms anisotropy component strongly suggests that a significant fraction of band 3 in both ghosts and intact cells is highly mobile and diffuses at the rate expected for a freely rotating dimer in the erythrocyte membrane.

The effects of two oppositely charged pyrene derivatives, 1-pyrenebutyrylcholine (PBC) and 1-pyrenebutyric acid (PBA), on red blood cell shape have been examined. Both compounds convert normal biconcave erythrocytes into echinocytes. However, with extended incubation time at elevated temperature, the morphology of PBC-induced echinocytes is reversed. Examination of probe uptake confirmed that, in contrast to PBA, equilibration of PBC with intact cells occurs very slowly. For PBA-induced echinocytes, it was possible to quantitate the fraction of probe bound in each half of the bilayer from nanosecond fluorescence measurements. Analysis of the heterogeneous decay showed that 71% of the bound PBA was associated with a lifetime (tau) of 102 ns and 29% with tau = 8 ns. It is likely that the later, highly quenched, component corresponds to fluorophores bound at the cytoplasmic surface because of efficient energy transfer to hemoglobin and that the long component corresponds to probe bound exclusively at the outer surface. Evidence in support of this interpretation was obtained by showing that when the paramagnetic cation Mn2+ bound at the extracellular surface the 102-ns component is quenched. The excimer fluorescence of PBC bound to red cells was examined and found to show time and temperature dependencies which correlate with morphological effects. These results indicate that red cells become crenated with PBC molecules are highly concentrated in the outer bilayer half and that shape reversal is subsequently brought about as PBC permeates and accumulates in the inner bilayer half. Finally, hemolysis protection due to PBC or PBA binding was observed also to show striking correlations with cell shape, In summary, these findings support the hypothesis [Sheetz, M. P., & Singer, S. J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4457] that shape changes are induced in red cells by amphiphilic molecules as a consequence of their relative partitioning between the two halves of the bilayer.