Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells.

beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.

Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

alpha 2-Macroglobulin bait region integrity. Role in determining fast-form structure.

To determine whether integrity of the bait region affects the structure of the remainder of human alpha 2-macroglobulin (alpha 2M), we have determined the separation between cysteine residues in a methylamine-reacted fast-form of alpha 2M. From reduction in fluorescence intensity of covalently-bound donor fluorophore caused by proximity to an acceptor, a separation of 35 +/- 8 A was calculated, which is identical to a previously determined value for proteinase-treated fast-form alpha 2M. This indicates that although bait region cleavage is the physiological route to conformational change in alpha 2M, bait region integrity per se does not significantly affect the structure of fast-form alpha 2M.

Simultaneous analysis of multiple fluorescence decay curves by Laplace transforms. Deconvolution with reference or excitation profiles.

The properties and potentials of the noniterative Laplace deconvolution (LAP2) (M. Ameloot and H. Hendrickx, Biophys. J. 44 (1983) 27) are further investigated. It is shown that LAP2 is exact and that no extrapolations have to be calculated or assumed for the data measured in the actual time window if the impulse response function of the investigated system can be described by a sum of exponentials. The formulas for the LAP2 deconvolution against the measured decay of a reference compound instead of the recorded excitation profile are derived. The procedure for the simultaneous analysis of multiple fluorescence decay curves by LAP2 is described in detail. This global analysis allows one to link any decay parameter, is fast and compares favorably with the nonlinear least-squares iterative reconvolution methods. Because of its short computation time the global analysis by LAP2 provides an efficient way to analyze the fluorescence decay surface in terms of decay associated spectra.

A second generation global analysis program for the recovery of complex inhomogeneous fluorescence decay kinetics.

A fluorescence spectroscopy global data analysis environment is described. Within this analysis environment, multidimensional fluorescence decay data (time and frequency domains) can be analyzed in terms of a wide variety of photophysical models. A generalized compartmental analysis structure is utilized, where one can specify the functions used to link the various compartments together. All fitting parameters may be characterized by either discrete or distributed values. Applications of these new analysis programs to the examination of phase transitions in lipid/membrane systems are described.

Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl.

Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated with Mal-ANS suggested that the inhibition of transport was due to a partial channel-blocking effect, and not to competition for the substrate binding site or to hindrance of carrier conformational change. In purified glucose carrier prepared from cells labeled on the exofacial sulfhydryl with Mal-ANS, a blue shift in the peak of fluorescence indicated that the fluorophore was in a relatively hydrophobic environment. Mal-ANS fluorescence in such preparations was quenched by ligands with affinity for the outward-facing carrier (ethylidene glucose, D-glucose, and maltose), but not by inhibitors considered to bind to the inward-facing carrier conformation (cytochalasin B or phenyl beta-D-glucoside). The effect of ethylidene glucose appeared to be related to an interaction with the glucose carrier, since the concentration dependence of ethylidene glucose-induced quench correlated well with the ability of the sugar analog to inhibit cytochalasin B binding to intact cells. The hydrophilic quenchers iodide and acrylamide decreased carrier-bound Mal-ANS fluorescence, resulting in downward-curving Stern-Volmer plots. Whereas ethylidene glucose enhanced iodide-induced quench, it had no effect on that of acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Fluorescence energy transfer is widely used for determination of intramolecular distances in macromolecules. The time dependence of the rate of energy transfer is a function of the donor/acceptor distance distribution and fluctuations between the various conformations which may occur during the lifetime of the excited state. Previous attempts to recover both distance distributions and segmental diffusion from time-resolved experiments have been unsuccessful due to the extreme correlation between fitting parameters. A method has been developed, based on global analysis of both donor and acceptor fluorescence decay curves, which overcomes this extreme cross-correlation and allows the parameters of the equilibrium distance distributions and intramolecular diffusion constants to be recovered with high statistical significance and accuracy. Simulation studies of typical intramolecular energy transfer experiments reveal that both static and dynamic conformational distribution information can thus be obtained at a single temperature and viscosity.

Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

Resolution of multiphasic reactions by the combination of fluorescence total-intensity and anisotropy stopped-flow kinetic experiments.

Multiphasic kinetics are often observed in stopped-flow investigations. To characterize further these kinetic phases, we have developed a methodology whereby fluorescence total intensity and anisotropy stopped-flow data can be combined in a single analysis. Fluorescence total intensity and anisotropy are highly interrelated and contain two very complementary forms of information. Total-intensity changes are useful in determining changes in populations with differing quantum yields, whereas anisotropy changes contain additional contributions caused by the rotational dynamics of the species. For cases in which the fluorescence quantum yield increases, the observed rate of anisotropy change will be more rapid than the total-intensity change, whereas in cases in which the total intensity decreases, the observed change in anisotropy will lag behind. In all cases, with quantum yield changes the stopped-flow anisotropy signals cannot be fit with models consisting of exponentials. Case studies examining these effects are described for the protein folding/refolding transitions of Staphylococcal nuclease and phosphoglycerate kinase. A multiphasic DNA exonuclease reaction using bacteriophage T4 DNA polymerase is also examined. In all of these cases, combined analysis of both data types revealed insights into reaction mechanism, which could not be obtained by either data type in isolation. Quantum yields and steady-state anisotropies associated with transiently populated intermediate species can be resolved. The data analysis methodologies described allow characterization of multiphasic reactions in terms of internally consistent kinetic rates, quantum yields, and steady-state anisotropies.

Analysis of binding in macromolecular complexes: a generalized numerical approach.

In this work, we introduce a generalized, global numerical methodology for analysis of binding phenomena in complex macromolecular assemblies. On the basis of a numerical algorithm (EQS) to solve systems of simultaneous free energy equations, binding profiles of simple to highly complex interacting systems can be analyzed over any concentration region without any need to generate an analytical form to describe the data. The output of the numerical algorithm is the concentration of each individual species in solution, allowing the generation of all possible binding profiles of the system (e.g., protein saturation by ligand). We present here the application of this approach to the DNA-protein subunit-ligand interactions of the trp repressor system as a typical example. From a practical point of view, the analysis program is capable of the rapid and simultaneous analysis of multiple binding profiles in terms of internally consistent sets of free energies. Given both the enormous complexity, as well as the underlying subtlety, involved in the regulation of biological function, the present generalized approach to analyzing macromolecular binding should find wide applications.

Mechanism of serpin action: evidence that C1 inhibitor functions as a suicide substrate.

Serpins form a family of structurally related proteins, many of which function in plasma as inhibitors of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation. To further characterize the mechanism by which serpins inhibit their target enzymes, we have studied the effect of temperature on the reaction of C1 inhibitor and the serine protease plasma kallikrein. At both 38 and 4 degrees C, C1 inhibitor (Mr 105,000) is cleaved by alpha-kallikrein (Mr 85,000 and 88,000) at position P1 (Arg444) of the reactive center, a reaction that leads to the formation of a covalent bimolecular enzyme-serpin complex (Mr 195,000) and cleaved but uncomplexed serpin (Mr 95,000). Between 38 and 4 degrees C, the product distribution is temperature-dependent, with more cleaved C1 inhibitor (Mr 95,000) formed at lower temperatures and correspondingly less Mr 195,000 complex. Studies employing intrinsic tryptophan fluorescence and 1H NMR spectroscopy show that this behavior is not caused by temperature-dependent conformational changes of kallikrein or C1 inhibitor. C1 inhibitor also behaves in this manner with the light chain of kallikrein and, to a lesser extent, with plasmin and C1s. These data are best explained by a branched reaction pathway, identical with the scheme describing the mechanism of action of suicide substrates. This scheme involves the formation of an enzyme-inhibitor intermediate, which can be stabilized into a covalent complex and/or dissociate into free enzyme and cleaved inhibitor, depending on the reaction conditions.

Conformational equilibrium of the reactive center loop of antithrombin examined by steady state and time-resolved fluorescence measurements: consequences for the mechanism of factor Xa inhibition by antithrombin-heparin complexes.

Activation of antithrombin by high-affinity heparin as an inhibitor of factor Xa has been ascribed to an allosteric switch between two conformations of the reactive center loop. However, we have previously shown that other, weaker binding, charged polysaccharides can give intermediate degrees of activation [Gettins, P. G. W., et al. (1993) Biochemistry 32, 8385-8389]. To examine whether such intermediate activation results from different reactive center loop conformations or, more simply, from a different equilibrium constant between the same two extreme conformations, we have used NBD covalently bound at the P1 position of an engineered R393C variant of antithrombin as a fluorescent reporter group and measured fluorescence lifetimes of the label in free antithrombin as well as in antithrombin saturated with long-chain high-affinity heparin, high-affinity heparin pentasaccharide, long-chain low-affinity heparin, and dextran sulfate. Steady state emission spectra, anisotropies, and dynamic quenching measurements were also recorded. We found that the large steady state fluorescence enhancements produced by binding of activators resulted from relief of a static quench of fluorescence of NBD in approximately 50% of the labeled antithrombin molecules rather than from any large change in lifetimes, and that similar lifetimes were found for NBD in all activated antithrombin-oligosaccharide complexes. Similar anisotropies and positions of the NBD emission maxima were also found in the absence and presence of activators. In addition, NBD was accessible to quenching agents in both the absence and presence of activators, with an at most 2-fold increase in quenching constants between these two extremes. The simplest interpretation of the partial static quench in the absence of activators, the different degrees of enhancement by different antithrombin activators, and the similar fluorescence properties and quenching behavior of the different states is that there are two distinct types of conformational equilibrium involving three distinct states of antithrombin, which we designate A, A', and B. A and A' represent low-affinity or inactive states of approximately equal energy, both having the hinge residues inserted into beta-sheet A. A is fluorescent, while A' is statically quenched. State B represents the activated loop-expelled conformation in which none of the NBD fluorophores are statically quenched, as a result of the loop, including the P1-NBD, moving away from the body of the antithrombin. Different activators are able to shift the equilibrium to the high-activity (B) state to different extents and hence give different degrees of measured activity, and different degrees of relief of static quench. The similar properties and accessibility of the NBD in the A and B conformations also indicate that the P1 side chain is not buried in the low-activity A conformation, suggesting that an earlier proposal that activation involves exposure of the P1 side chain cannot be the explanation for activation. As an alternative explanation, heparin activation may give access to an exosite on antithrombin for binding to factor Xa and hence be the principal basis for enhancement of the rate of inhibition.

Orientational distribution of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles as determined by global analysis of frequency domain fluorimetry data.

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.

Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.

Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the alpha/beta type. Extensive domain-domain interactions involve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located alpha-helix. In order to monitor spectroscopically the conformational changes that occur in the individual domains and at the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants. In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333) [Szpikowska, B. K., Beechem, J. M., Sherman, M. A., & Mas, M. T. (1994) Biochemistry 33, 2217-2225], four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122). The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence. These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper. During the first transition (Cm approximately 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact but lacks much of the tertiary structure that characterizes the native state. A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain. Complete unfolding of the N-terminal domain occurs during the second transition (Cm approximately 0.9 M).

Sequential domain unfolding in phosphoglycerate kinase: fluorescence intensity and anisotropy stopped-flow kinetics of several tryptophan mutants.

Stopped-flow total intensity and anisotropy experiments on single tryptophan containing mutants of yeast phosphoglycerate kinase (PGK) located in either the carboxy-terminal domain (W308 and W333), amino-terminal domain (W48 and W122), or "hinge" region (W194 and W399) were performed. The results obtained for single tryptophans in individual domains suggest that the unfolding of PGK by guanidinium hydrochloride is a sequential process in which unfolding of the carboxy-terminal domain is followed by the unfolding of the amino-terminal domain. A kinetic intermediate has been detected which consists of an unfolded carboxy-terminal domain and an altered amino-terminal domain, identical in hydrodynamic properties with the native state, but hyperfluorescent. In contrast to the C-terminal tryptophans, which exhibit concurrent total intensity and anisotropy changes in the entire denaturant concentration range (0-->2 M), the N-terminal tryptophans experience a large increase in fluorescence intensity and a constant anisotropic environment at low concentrations of denaturant, corresponding to the first transition region of the equilibrium unfolding profile. Anisotropy changes for the N-terminal probes are observed above 1 M Gdn-HCl, the region corresponding to the second equilibrium unfolding transition. Stopped-flow experiments performed on PGK mutants with two tryptophans (i.e., with a single tryptophan in each domain) confirm that each domain unfolds independently, and that the individual site-specific mutations do not significantly alter the unfolding pathway. Unfolding kinetics experiments with tryptophans situated in the hinge reveal that the region sensed by W399 unfolds before the carboxy-terminal domain, whereas W194 senses unfolding of both domains.

The interaction of epidermal growth factor with its receptor in A431 cell membranes: a stopped-flow fluorescence anisotropy study.

We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescence anisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with the EGF receptor over a wide range of FITC-EGF concentrations, membrane dilutions, and time scales (milliseconds to minutes). Fluorescence anisotropy-based equilibrium binding titrations were also performed. All studies utilized the same receptor preparation, ligand preparation, and detection system. The entire data surface (approximately 78,000 data points) was simultaneously analyzed using global analysis techniques with a variety of kinetic models. Our analysis identified with a high level of confidence receptor populations with two association rate constants (k(on) = 1.2 x 10(6) M-1 s-1, 7.2 x 10(6) M-1 s-1) and three dissociation rate constants (k(off0 = 0.95 x 10(-2) s-1, 0.13 x 10(-2) s-1, 0.32 x 10(-3) s-1), which reflect the presence of at least two distinct receptor populations in A431 cell membranes. Analysis of the kinetic data was found to be much more sensitive to the presence of multiple receptor populations than was the analysis of the equilibrium binding data.