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Dynamic behavior during the drying process of phospholipid multilayers composed of 1,2-dipalmitoyl- sn - glycero -3-phosphocholine and 1,2-dihexadecanoyl- sn -glycero-3-phospho-(1′-rac-glycerol) in mixtures of 1:4, was investigated by means of attenuated total reflection (ATR) Fourier transform infrared spectroscopy (FTIR). The interaction of the antimicrobial peptide halictine-1 (HAL-1), a linear dodecapeptide isolated from the venom of eusocial bee Halictus sexcinctus , with the lipid layer and its influence on the dynamics of the lipid layer was also studied. Analysis of ATR-FTIR spectra of the drying process by principal component analysis (PCA) clearly showed the sensitivity of C O vibrations at 1737 cm −1 , PO 2 vibrations in the region 1000–1250 cm −1 and CH stretching vibrations at 2850 and 2950 cm −1 to the hydration of the lipid layer. Nevertheless, PCA revealed that the lipid layers periodically oscillate between dehydration/hydration states. The protective influence of HAL-1 on the lipid layer, including the disappearance of dehydration/hydration oscillations, and slowing down of the drying of the system in the presence of the peptide was observed. PCA indicates a two-stage process of the interaction of HAL-1 with lipid layers and as well as the influence of HAL-1 on vibrations of the C O and PO 2 of lipid groups, whereas CH 2 and CH 3 vibrations remain intact during the binding of the peptide. The peptide binds to phospholipid head groups, changes its structure from a random coil to an α-helix structure, and interacts with the C O groups of the lipids, staying on the surface of the membrane. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S–S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S–S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions. [ABSTRACT FROM AUTHOR]

The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE. [ABSTRACT FROM AUTHOR]

RNase L, a key enzyme in the host defense system, is activated by the binding of 2′–5′-linked oligoadenylates (2–5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2–5 pA 4 and three tetramers with 3′-end AMP units replaced with ribo- , arabino- and xylo- configured phosphonate analogs of AMP (pA 3 X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α–β structural switch may fix domain positions in the hinge region (residues ca. 336–363) during homodimer formation. [ABSTRACT FROM AUTHOR]

Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable. [ABSTRACT FROM AUTHOR]