Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

A calorimetric examination of the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes

The effect of myotoxin a on the thermotropic phase behavior of aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was examined using differential scanning calorimetry (DSC). Myotoxin a significantly altered the normal phase behavior of DMPC in a concentration dependent fashion. This effect is perturbed by Ca2+ and is sensitive to ionic strength and pH. High concentrations of toxin eliminate the characteristic pretransition associated with the polar head group of DMPC. They also increase the temperature of the main gel-to-liquid crystal transition from 23 degrees C to 32-35 degrees C. At low concentrations of toxin, the first visible effect is upon the pretransition which is split into two components that diminish with time. The main transition is less affected at low toxin concentrations, although the magnitude of the transition is reduced while it is simultaneously shifted to higher temperatures. The main transition is also split into multiple components. The toxin also had pH specific effects on the phase behavior of DMPS. Above physiological pH (8.5) the normal transition of DMPS at 36-38 degrees C was split in the presence of myotoxin a and new components appeared centered at 31 degrees C and 35 degrees C. These observations are consistent with reports that the skeletal muscle membrane system is the major site of the myonecrotic effect of myotoxin a.     

Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.

The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.     

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

A differential scanning calorimetric study of the bovine lens crystallins

Differential scanning calorimetry was performed on the five major lens crystallin fractions [HM-alpha, alpha, beta H, beta L, and (beta s + gamma)] of the bovine lens as well as on more purified forms of alpha- and gamma-crystallins. All were found to be relatively thermally stable although the alpha-crystallin were found to at least partially unfold at an approximately 10 degrees C lower temperature than the beta and gamma fractions. Increasing protein concentration had little effect on gamma-crystallin thermograms but had marked effects on those of the alpha- and beta-crystallins. Increases in the thermal stability with increasing protein concentration for the beta-crystallins can be explained most simply by the known beta L/beta H equilibrium, but, in the case of the alpha-crystallins, excluded volume effects may be an important factor. In both cases, the increased stability at high concentrations could be of physiological relevance. As well as the expected endothermic unfolding transitions, all of the lens crystallins revealed exothermic peaks that correlate with protein precipitation. Interestingly, this phenomenon occurs only after extensive structural alteration in the case of the alpha-crystallins but is present very early in the initial stages of structural perturbation of the beta- and gamma-crystallins.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein. We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n‐octyl‐oligo‐oxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,N‐dimethyldodecylamine N‐oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size‐dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.     

Conformational origin of the aggregation of recombinant human factor VIII

Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.     

Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri

Shigella flexneri causes a self-limiting gastroenteritis in humans, characterized by severe localized inflammation and ulceration of the colonic mucosa. Shigellosis most often targets young children in underdeveloped countries. Invasion plasmid antigen C (IpaC) has been identified as the primary effector protein for Shigella invasion of epithelial cells. Although an initial model of IpaC function has been developed, no detailed structural information is available that could assist in a better understanding of the molecular basis for its interactions with the host cytoskeleton and phospholipid membrane. We have therefore initiated structural studies of IpaC, IpaC I', (residues 101-363 deleted), and IpaC Delta H (residues 63-170 deleted). The secondary and tertiary structure of the protein was examined as a function of temperature, employing circular dichroism and high resolution derivative absorbance techniques. ANS (8-anilino-1-napthalene sulfonic acid) was used to probe the exposure of the hydrophobic surfaces under different conditions. The interaction of IpaC and these mutants with a liposome model (liposomes with entrapped fluorescein) was also examined. Domain III (residues 261-363) was studied using linker-scanning mutagenesis. It was shown that domain III contains periodic, sequence-dependent activity, suggesting helical structure in this section of the protein. In addition to these structural studies, investigation into the actin nucleation properties of IpaC was conducted, and actin nucleation by IpaC and some of the mutants was exhibited. Structure-function relationships of IpaC are discussed.     

Dietary input of microbes and host genetic variation shape among-population differences in stickleback gut microbiota

To explain differences in gut microbial communities we must determine how processes regulating microbial community assembly (colonization, persistence) differ among hosts and affect microbiota composition. We surveyed the gut microbiota of threespine stickleback (Gasterosteus aculeatus) from 10 geographically clustered populations and sequenced environmental samples to track potential colonizing microbes and quantify the effects of host environment and genotype. Gut microbiota composition and diversity varied among populations. These among-population differences were associated with multiple covarying ecological variables: habitat type (lake, stream, estuary), lake geomorphology and food- (but not water-) associated microbiota. Fish genotype also covaried with gut microbiota composition; more genetically divergent populations exhibited more divergent gut microbiota. Our results suggest that population level differences in stickleback gut microbiota may depend more on internal sorting processes (host genotype) than on colonization processes (transient environmental effects).