Fen-Ni Fu

Secondary Structure Estimation of Proteins Using the Amide III Region of Fourier Transform Infrared Spectroscopy: Application to Analyze Calcium-Binding-Induced Structural Changes in Calsequestrin

A Fourier transform infrared spectroscopic method has been developed to analyze protein secondary structure by employing the amide III spectral region (1350–1200 cm−1)· Benefits of using the amide III region have been shown to be substantial. The interference from the water vibration (∼1640 cm−1) in the amide I region can be avoided when one is using the amide III band; furthermore, the amide III region also presents a more characterized spectral feature which provides easily resolved and better defined bands for quantitative analysis. Estimates of secondary structure are accomplished with the use of Fourier self-deconvolution, second derivatization, and curve-fitting on original protein spectra. The secondary structure frequency windows (α-helix, 1328–1289 cm−1; unordered, 1288–1256 cm−1; and β-sheets, 1255–1224 cm−1) have been obtained, and estimates of secondary structural contents are consistent with X-ray crystallography data for model proteins and parallel results obtained with the use of the amide I region. We have further applied the analysis to the structural change of calsequestrin upon Ca2+ binding. Treatment of calsequestrin with 1 mM Ca2+ results in the formation of crystalline aggregates accompanied by a 10% increase in α-helical structure, which is consistent with previous results obtained by Raman spectroscopy. Thus the amide III region of protein IR spectra appears to be a valuable tool in estimating individual protein secondary structural contents.

A Protease-Resistant Novel Hemagglutinin Purified from Type A Clostridium botulinum

Botulinum neurotoxin is a food poisoning agent produced by Clostridium botulinum. The neurotoxin is a 150-kDa protein that causes flaccid muscle paralysis by blocking neurotransmitter release at neuromuscular junctions. The neurotoxin is produced along with a group of neurotoxin associated proteins (NAPs), which protect it from the low pH and proteases of the gastrointestinal (GI) tract. We have isolated, for the first time, one of the major components of NAPs in a pure form. The isolated protein is a 33-kDa single polypeptide (Hn-33) that exhibits hemagglutination activity. Specific polyclonal antibodies against the Hn-33 are able to block the hemagglutination activity of the neurotoxin complex, which indicates that perhaps Hn-33 is the only strong hemagglutinating protein in the complex. The Hn-33 was found be resistant to trypsin and other protease digestion, a feature that could play a role in the protection of the neurotoxin in the GI tract during its toxicoinfection.

Molecular Properties of a Hemagglutinin Purified from Type A Clostridium botulinum

Clostridium botulinum causes the food poisoning disease botulism by producing botulinum neurotoxin, the most potent toxin known. The neurotoxin is produced along with a group of neurotoxin-associated proteins, or NAPs, which protect it from the low pH and proteases of the gastrointestinal tract. Recently, we isolated one of the major components of NAPs, a 33-kDa hemagglutinin (Hn-33) [Fu et al. (1998), J. Protein Chem.17, 53–60]. In this study, we present molecular properties of Hn-33 derived from several biochemical and biophysical techniques. Hn-33 in pure form requires a 66-fold lower concentration of sugar inhibition of its hemagglutination activity than in its complexed form with the neurotoxin and other NAPs. However, its protease resistance is not affected by sugar binding. Based on FT-IR and circular dichroism (CD) analysis, Hn-33 is a predominantly β-sheet protein (74–77%). Hn-33 analysis by laser desorption mass spectrometry and size exclusion column chromatography reveals that it exists predominantly in a dimeric form in the aqueous solution. Even a very low concentration of SDS (0.05%) irreversibly destroyed the biological activity of Hn-33 by changing its secondary structure as revealed by far-UV CD analysis.

Use of Fourier Transform Infrared/Attenuated Total Reflectance Spectroscopy for the Study of Surface Adsorption of Proteins

The adsorption behavior of lysozyme and immunoglobulin G (IgG) onto a ZnSe crystal surface has been detected by the FT-IR/ATR technique. With this technique we are able to detect the protein adsorption process at a very low concentration (0.0005 mg/mL). The equation of Sperline et al. [Langmuir 3, 198 (1987)] was successfully applied to calculate the adsorption density of protein from an aqueous solution on a real-time basis. The monolayer formation of lysozyme was observed at a concentration range of 10−7 M to 10−5 M. A monolayer-to-multilayer adsorption transition of lysozyme was clearly observed from the adsorption isotherm plot at 35 μM. A differential adsorption density was observed for IgG that could be explained on the basis of its differential size and surface charge.

Spectroscopic analysis of low pH and lipid-induced structural changes in type A botulinum neurotoxin relevant to membrane channel formation and translocation.

Botulinum neurotoxin (BoNT) is an extremely toxic protein to animals and humans. In its mode of action, one of its subunits mediates its translocation by integrating itself into the membrane bilayer. We have examined the membrane channel activity of type A BoNT (BoNT/A) and its heavy (H) chain in planar lipid membrane under various pH conditions to understand the possible role of the channel activity in the translocation of the BoNT/A light (L) chain under physiological conditions. Only BoNT/A H chain, and not the BoNT/A, exhibited membrane channel activity for translocation of ions. The H chain-induced increase in conductance did not require a pH gradient across the lipid membrane, although it was enhanced by a pH gradient. To understand the molecular basis of the membrane channel activity and the translocation of the L chain, the secondary structure of BoNT/A and its H and L chains were analyzed using circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy at different pH values. BoNT/A showed no structural alternation upon acidifying the buffer pH. However, an increase in beta-sheet content of BoNT/A H chain at low pH was noted when examined by FT-IR. The L chain structure significantly changed with decrease in pH, and the change was mostly reversible. In addition, the neurotoxin and its subunit chains induced a partially reversible aggregation of liposomes at low pH, which indicated their integration into the lipid bilayer. Temperature-induced denaturation studies of BoNT/A H chain indicated major structural reorganization upon its interaction with membrane, especially at low pH.

Calcein Permeability of Liposomes Mediated by Type A Botulinum Neurotoxin and Its Light and Heavy Chains

The mode of botulinum neurotoxin action involves binding of its heavy chain for internalization into the presynaptic end of a nerve cell through endocytosis. The low-pH conditions of endosomes trigger translocation of the light chain across the endosomal membrane to the cytosol, where the light chain cleaves specific target proteins involved in the docking and fusion of synaptic vesicles for acetylcholine release. In an effort to model the interaction of botulinum neurotoxin and its subunit chains with lipid bilayer at low pH during the translocation process, we have examined type A botulinum neurotoxin-mediated calcein release from asolectin liposomes. At equimolar concentration (0.1 μM), the neurotoxin and its heavy and light chains evoked 23%, 58%, and 28% calcein release, respectively. Calcein release was observed only when the cis-side (the side to which neurotoxin samples were added) pH was lowered to 4. Calcein release activity of the heavy chain was mostly blocked (76%) by a polyclonal antibody raised against the neurotoxin. Additionally, two peptide-specific polyclonal antibodies derived from the N-terminal and C-terminal halves of the heavy chain were also able to block the calcein release activity by 15–20%. In summary, these results suggest that calcein release from liposomes is specifically mediated by the heavy chain, and the light chain also integrates into the membrane. Implications of these results for the molecular mode of neurotoxin light-chain translocation across the endosomal membrane are discussed.

Fourier transform infrared analysis of amide III bands of proteins for the secondary structure estimation

Protein secondary structure has been analyzed using a Fourier transform infrared spectroscopic method in the amide III region. Although extensive work has been done on protein secondary structure using the amide I region (1700 - 1600 cm-1), the amide III region has not been utilized in the past for its potential in protein structural analysis. One of the major reasons for non-use of the amide III vibrations is perhaps the very weak signal in the amide III frequency region (1200 - 1350 cm-1). However, benefits of using the amide III region are substantial. For example, water vibrations do not interfere with the protein spectrum unlike in the amide I region. In the amide III region, the protein spectrum is better resolved into individual bands than in the amide I region. This feature allows for a greater ease in peak definement of the protein spectra. In the amide III region, the bands for the individual secondary structures ((alpha) -helix, (beta) -sheet and random coils) do not overlap as much as they do in the amide I region. This lack of overlapping allows for easier and a more reliable means of peak assignment, and secondary structure band positions are easier to determine. Amide III region of protein IR spectra appears to be a valuable tool in estimating the amount of secondary structure present in proteins.

Fourier Transform Infrared Spectroscopic Analysis of Proteins in Terms of Detectability, Conformation and Surface Adsorption Density

Publisher Summary Spectroscopic methods commonly used to analyze the polypeptide folding of proteins include fluorescence, circular dichroism, Fourier transform infrared (FT-IR), Raman, and nuclear magnetic resonance spectroscopies. This chapter discusses the use of attenuated total reflectance (ATR) technique for sample handling to test the limits of the sensitivity of FT-IR spectroscopy for protein structural analysis. The use of FT-IR-ATR for the conformational analysis of low concentration proteins can be extremely important to biochemists and molecular biologists who have a minute amount of proteins purified or who have produced small amounts of proteins by genetic engineering. For proteins or protein fragments that are not readily soluble in aqueous solutions, the FT-IR1-ATR approach may provide a useful means to analyze their structure. The technique should also provide a way to analyze the structure of proteins at higher concentrations, which is not possible with circular dichroism or fluorescence. A comparison of protein structure at high concentration such as in precipitate on crystals, and high concentration aqueous solution versus low concentration aqueous solution may provide information about the relationship between structure and biological activity. FT-IR-ATR spectroscopy of polypeptides has a high potential for their conformational analysis at concentration levels that cannot be accurately analyzed by commonly used techniques such as circular dichroism or intrinsic fluorescence.

Gene probe-based detection of type E botulinum neurotoxin binding protein using polymerase chain reaction

Using primers based on the nucleotide sequence of a neurotoxin binding protein from Type E Clostridium botulinum cultures, an amplified DNA product was obtained through polymerase chain reaction. The 400 base pair amplified DNA fragment was detectable with as low as 0.1 pg template DNA from Type E C. botulinum, and its fidelity was confirmed by Southern blotting using a DNA probe designed to detect the expected amplified DNA fragment. On the other hand, no DNA amplification was observed with as high as 10 ng template DNA from related Types A and B C. botulinum or from C. tetani, indicating the specificity of the probe.