Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an SN2 displacement reaction

The semiempirical PM3 method, calibrated against ab initio HF/6–31+G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO− with DCE; (ii) the structures of the transition states in the gas-phase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal/mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Suppression of HIV replication by lymphoid tissue CD8+ cells correlates with the clinical state of HIV-infected individuals

Lymphoid tissues from asymptomatic HIV-infected individuals, as compared with symptomatic HIV-infected subjects, show limited histopathological changes and lower levels of HIV expression. In this report we correlate the control of HIV replication in lymph nodes to the non-cytolytic anti-HIV activity of lymphoid tissue CD8+ cells. Five subjects at different stages of HIV-related disease were studied and the ability of their CD8+ cells, isolated from both lymphoid tissue and peripheral blood, to inhibit HIV replication was compared. CD8+ cells from lymphoid tissue and peripheral blood of two HIV-infected long-term survivors suppressed HIV replication at a low CD8+:CD4+ cell ratio of 0.1. The CD8+ cells from the lymphoid tissue of a third asymptomatic subject suppressed HIV replication at a CD8+:CD4+ cell ratio of 0.25; the subject’s peripheral blood CD8+ cells showed this antiviral response at a lower ratio of 0.05. The lymphoid tissue CD8+ cells from two AIDS patients were not able to suppress HIV replication, and the peripheral blood CD8+ cells of only one of them suppressed HIV replication. The plasma viremia, cellular HIV load as well as the extent of pathology and virus expression in the lymphoid tissue of the two long-term survivors, were reduced compared with these parameters in the three other subjects. The data suggest that the extent of anti-HIV activity by CD8+ cells from lymphoid tissue relative to peripheral blood correlates best with the clinical state measured by lymphoid tissue pathology and HIV burden in lymphoid tissues and blood. The results add further emphasis to the importance of this cellular immune response in controlling HIV pathogenesis.

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN↓NGGCC, where N is any base and ↓ is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SfiI, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SfiI cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SfiI cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for kcat and Km were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SfiI activity is thus highly dependent on conformational variations in the spacer DNA.

Non-Arrhenius kinetics for the loop closure of a DNA hairpin

Intramolecular chain diffusion is an elementary process in the conformational fluctuations of the DNA hairpin-loop. We have studied the temperature and viscosity dependence of a model DNA hairpin-loop by FRET (fluorescence resonance energy transfer) fluctuation spectroscopy (FRETfs). Apparent thermodynamic parameters were obtained by analyzing the correlation amplitude through a two-state model and are consistent with steady-state fluorescence measurements. The kinetics of closing the loop show non-Arrhenius behavior, in agreement with theoretical prediction and other experimental measurements on peptide folding. The fluctuation rates show a fractional power dependence (β = 0.83) on the solution viscosity. A much slower intrachain diffusion coefficient in comparison to that of polypeptides was derived based on the first passage time theory of SSS [Szabo, A., Schulten, K. & Schulten, Z. (1980) J. Chem. Phys. 72, 4350–4357], suggesting that intrachain interactions, especially stacking interaction in the loop, might increase the roughness of the free energy surface of the DNA hairpin-loop.

Thermal unfolding of human high-density apolipoprotein A-1: implications for a lipid-free molten globular state.

Apolipoprotein A-1 (apoA-1) in complex with high-density lipoprotein is critically involved in the transport and metabolism of cholesterol and in the pathogenesis of atherosclerosis. We reexamined the thermal unfolding of lipid-free apoA-1 in low-salt solution at pH approximately 7, by using differential scanning calorimetry and circular dichroism. At protein concentrations <5 mg/ml, thermal unfolding of apoA-1 is resolved as an extended peak (25 degrees C-90 degrees C) that can be largely accounted for by a single reversible non-two-state transition with midpoint Tm 57 +/- 1 degree C, calorimetric enthalpy deltaH(Tm)= 200 +/- 20 kcal/mol (1 kcal = 4.18 kJ), van't Hoff enthalpy deltaHv(Tm) approximately 32.5 kcal/mol, and cooperativity deltaHv(Tm)/deltaH(Tm) approximately 0.16. The enthalpy deltaH(Tm) can be accounted for by melting of the alpha-helical structure that is inferred by CD to constitute approximately 60% of apoA-1 amino acids. Farand near-UV CD spectra reveal noncoincident melting of the secondary and tertiary structural elements and indicate a well-defined secondary structure but a largely melted tertiary structure for apoA-1 at approximately 37 degrees C and pH 7. This suggests a molten globular-like state for lipid-free apoA-1 under near-physiological conditions. Our results suggest that in vivo lipid binding by apoA-1 may be mediated via the molten globular apolipoprotein state in plasma.