C. Russell Middaugh

Molecular mechanism of spider silk elasticity

Spider major ampullate (drag-line) silk is an extracellular fibrous protein which has impressive characteristics of strength and elasticity. This silk has been hypothesized to predominantly consist of a single protein, containing regions of antiparallel beta-sheets which are interspersed with amorphous segments responsible for its elastic properties. A rubber-like mechanism has been suggested to account for this elasticity, but the specific molecular mechanism is unknown. Using Fourier transform infrared spectroscopy (FTIR) we found evidence of either helix formation or reorientation of preexisting helices when axial tension is applied to the spider silk fiber. CD studies of a peptide derived from the silk gene repeat sequence show that it can form beta-sheets at high temperatures while alpha-helices are induced in 2,2,2-trifluoroethanol. These results suggest a possible molecular mechanism for the elasticity of spider silk fibers. It is proposed that the elastic process involves the formation and disruption of alpha-helical Ala-rich regions which are interspersed among stable beta-sheet domains.

Chemical crosslinking of cell membranes

SummaryThe complexity of cell membranes makes the resolution of their macromolecular topology one of the more challenging problems in modern molecular and cellular biochemistry. Despite the difficulties inherent in any such analysis, a surprisingly simple yet powerful approach exists that has consistently yielded valuable results. This method is chemical crosslinking, in which cell membranes are treated with crosslinking reagents (usually bifunctional) which produce covalent linkages between membrane components. The resultant complexes are usually then separated and identified by electrophoresis. This review is intended to provide a guide to the i.nvestigator who is unfamiliar with this approach. The overall strategy of crosslinking is discussed including selection of reagents, conditions to optimize crosslinking and the cleavage of crosslinked complexes to regenerate the original target for identification purposes. The crosslinking of biological membranes is then reviewed with special emphasis on recent advances including macromolecular photoaffinity labeling, kinetic analysis to probe symmetry properties and potential artifacts that may complicate interpretation of results. Examples of specific applications of crosslinking to membranes are presented in tabular form. The final portion of the review discusses the synthesis and properties of the most widely employed crosslinking reagents. Available reagents are summarized in a series of comprehensive tables. It is hoped that our discussion will provide the uninitiated investigator with sufficient information to ascertain the applicability of chemical crosslinking to particular areas of interest.

Near-infrared photoacoustic spectroscopy of proteins

A major problem encountered with the use of electronic spectroscopy in the analysis of biological materials in the ultraviolet, visible, and infrared region involves the limited range of the physical state of samples that can be examined. In an attempt to expand this range, photoacoustic spectra of both solid- and solution-state proteins have been obtained in the near-infrared region. Solid proteins generate detailed spectra in the region 1.0-2.6 micron, resulting primarily from hydrogenic overtone and combinational modes. Harmonics and combinations of amide group frequencies which display significant spectral complexity are observed between 1.4 and 1.7 micron, although they appear to manifest only limited conformational sensitivity. Solution spectra in D2O are of much lower resolution. Assignments of peaks for both solution- and solid-state proteins are presented and the advantages and disadvantages of the use of near-infrared photoacoustic spectroscopy with proteins are discussed.

Analysis of protein association by partitioning in aqueous two-phase polymer systems: Applications to the tetramer-dimer dissociation of hemoglobin

Abstract A new simple and rapid method for the determination of protein-protein association constants is described. By maximizing experimental conditions in which size becomes the controlling variable, analysis of the effect of protein concentration on the partitioning behavior of proteins in aqueous two-phase polymer systems permits an accurate estimate of protein association constants. When employed to investigate the tetramer-dimer dissociation of human oxy- and methemoglobin in the presence and absence of high salt concentration, values for the dissociation constant are obtained that are consistent with those obtained by other methods.

Kinetics of the precipitation of cryoimmunoglobulins

The kinetics of the cryoprecipitation of two monoclonal IgG and two monoclonal IgM cryoimmunoglobulins, two IgM/IgG mixed cryoglobulins and a series of cold soluble monoclonal IgG and IgM immunoglobulins in the presence of polyethylene glycol have been compared by time dependent turbidity measurements. The effects of temp and ionic strength on kinetic processes are described in detail. The monoclonal cryoimmunoglobulins display lag times which are not seen with the other proteins, suggesting a critical nucleation event. The protein concn dependence of the lag times indicate that these nucleation centers contain only a few immunoglobulin molecules. Direct evidence for the existence of precipitation nuclei was obtained from dynamic light scattering studies of two of the monoclonal proteins during their lag periods. Both proteins manifested an approx. 20% decrease in their mean diffusion coefficients (corresponding to a 25% increase in Stokes radius) prior to detectable precipitation. This suggests the formation of nuclei between 2 and 8 times the size of the monomeric proteins. It is postulated that the increasing size of mixed cryoglobulin complexes with decreasing temp provides analogous nucleation sites. The latter stages of precipitation appear to be kinetically similar for all proteins examined, although the size and shape of the aggregates are quite variable.

Does random collisional cross-linking occur?

In fluid membranes, mobile molecules are thought to collide at high frequencies. Concern has been expressed as to whether these colliding molecules are cross-linking during the chemical cross-linking of membrane molecules, thereby creating problems in interpreting such experiments. Hemoglobin was used as a model to test this possibility. Oligomers larger than the tetramer could be cross-linked depending on factors such as hemoglobin concentration, duration of the cross-linking reaction and the type of reagent. Under certain conditions, however, such as a hemoglobin concentration less than 150 microM or a duration of cross-linking shorter than 15 min, larger oligomers were not detectable. Analysis of these data suggests that the probability of random collisional cross-links under normal conditions is insignificant.

Comparative spectroscopic studies of four crotoxin homologs and their subunits

Structures of four related neurotoxins and their purified subunits from the venoms of Crotalus durissus terrificus, C. vegrandis, C. s. scutulatus and C. viridis concolor were examined by circular dichroism (CD), deconvolution Fourier-transform infrared (FTIR) and fluorescence spectroscopy. CD spectra suggest that in general, the isolated subunits were decreased slightly in alpha-helix, while they were increased in beta-sheet structure, relative to intact toxins. These results were consistent with FTIR results. Fluorescence quenching (50-80%) was also observed in three of the four intact toxins as compared to spectra predicted by summation of free acidic and basic subunit spectra. It was tempting to conclude from these results that major conformational changes occur in individual subunits upon formation of the dimeric toxins. Intact crotoxin, however, when exposed to urea, yields spectra (CD, FTIR and fluorescence) that are virtually identical to control intact crotoxin. These findings suggest that the enhanced fluorescence exhibited by the isolated subunits, as well as the secondary structural changes in alpha-helix and beta-sheet, are artifacts resulting from irreversible structural changes that occur during subunit isolation by urea ion-exchange chromatography. In spite of these structural changes, LD50 values of intact crotoxin reassembled from isolated subunits are unaltered from those of native crotoxin.

Spectroscopic characterization of textilotoxin, a presynaptic neurotoxin from the venom of the Australian eastern brown snake (Pseudonaja t. textilis)

Spectroscopic behavior of textilotoxin, from the venom of Pseudonaja t. textilis, and its subunits were investigated using fluorescence, circular dichroism and Fourier transform infrared spectroscopy. Circular dichroism spectra of the B, C and D subunits indicate considerable similarity in their alpha-helix and beta-sheet contents. By contrast, the A subunit displays significantly more beta-sheet and remainder structure. FTIR spectra confirm conclusions drawn from CD spectra. Fluorescence spectra indicate that, in general, tryptophan residues in the A, B and D subunits are relatively exposed to the solvent. The C subunit exhibits no fluorescence, suggesting a lack of tryptophan. Comparisons of individual subunit spectra with those of the intact toxin suggest that significant changes in secondary structure may occur when the toxin dissociates.

Thermodynamics of Monoclonal and Mixed Cryoimmunoglobin Solubilization

The direct calorimetric determination of heats of solution for four monoclonal and three mixed (IgM/IgG) cryoglobulins is described. Values obtained by differential scanning calorimetry (DSC) are compared to values of the apparent delta Hsol obtained by a polyethylene glycol (PEG) precipitation method. The four monoclonal cryoglobulins manifest heats of solution determined by DSC to be of the same order of magnitude as heats obtained by PEG precipitation, although DSC values were 25 to 125% lower than the corresponding vant Hoff enthalpies. Values of delta Hsol for mixed cryoglobulins were significantly greater than monoclonal cryoglobulins on a molar basis. These higher values are primarily attributed to the greater surface area of these complexes which results in more extensive contact between molecules in the solid phase. No evidence was found that conformational changes contributed to the calorimetric delta Hsol values employing a variety of spectroscopic methods.

Is the Tyrosine Rich Eggshell Protein of Schistosoma Mansoni an Electron Transport Chain

The schistosome eggshell is composed of two kinds of cross-linked proteins. One is glycine rich and the other is extremely tyrosine rich and highly repetitive. The highly conserved sequence of the tyrosine residues in the tyrosine rich protein suggests that it may play a role as an electron transport chain during eggshell cross-linking. Studies using model synthetic peptides suggest that the tyrosine rich protein may adopt a left-handed structure, possibly a left-handed alpha-helix.