Search

The solution structure of the cold-shock-like protein from R. rickettsii, the causative agent of Rocky Mountain spotted fever, is reported., Rocky Mountain spotted fever is caused by Rickettsia rickettsii infection. R. rickettsii can be transmitted to mammals, including humans, through the bite of an infected hard-bodied tick of the family Ixodidae. Since the R. rickettsii genome contains only one cold-shock-like protein and given the essential nature of cold-shock proteins in other bacteria, the structure of the cold-shock-like protein from R. rickettsii was investigated. With the exception of a short α-helix found between β-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded β-barrel structure found in most cold-shock domains. Additionally, the R. rickettsii cold-shock-like protein, with a ΔG of unfolding of 18.4 kJ mol−1, has a similar stability when compared with other bacterial cold-shock proteins.

Advanced chemistry students are introduced to surface-enhanced resonance Raman scattering (SERRS) by studying how sodium copper chlorophyllin (CuChl) adsorbs onto silver colloids (CuChl/Ag) as a function of pH. Using both SERRS and visible extinction spectroscopy, the extent of CuChl adsorption and colloidal aggregation are monitored. Initially at pH ∼10, the colloids possess an overall negative surface charge, preventing attraction. Surface-enhanced Raman scattering (SERS) is not detected at that pH because the relatively small colloids do not possess a surface plasmon in resonance with the laser excitation frequency (633 nm). Once the solution pH decreases slightly, colloidal clusters form and SERRS is detected. As the pH is lowered to a value of 4 or 3, protonation of CuChl results in colloidal aggregation and precipitation. If adsorption and aggregation are halted by adding H2SO4(aq) and then NaOH(aq), an intense SERRS signal is steadily detected from acidic pH (about 4), back to the initial basic pH (about 10).

The bond dissociation energy for loss of C2H4 from Fe(CO)3(C2H4)2, produced by the reaction of C2H4 + Fe(CO)3(C2H4), has been determined as 21.3 ± 2.0 kcal/mol. An estimate is made for a lower limit for the bond dissociation energy of Fe(CO)4(C2H4), which can be formed by reaction of CO + Fe(CO)3(C2H4) or Fe(CO)4 + C2H4 with rate constants of (4.3 ± 0.8) × 10-12 and (1.7 ± 0.2) × 10-13 cm3/(molecule s) at 24 °C, respectively. The values for these bond dissociation energies are compared with those determined in prior studies of these systems. A new compound with infrared absorptions at 2147, 2091, and 2068 cm-1 is best assigned as Fe(CO)3(C2F4)2. A rate constant of (5.4 ± 1.7) × 10-12 cm3/(molecule s) at 24 °C is reported for the reaction of C2F4 with Fe(CO)3(C2F4) to form Fe(CO)3(C2F4)2. Fe(CO)4(C2F4) can be formed by reaction of C2F4 and Fe(CO)4, with a rate constant of (1.8 ± 0.4) × 10-14 cm3/(molecule s) at 24 °C. Infrared absorptions observed at 2135, 2074, and 2043 cm-1 are assigned to this species. ...

Rate constants for the reactions of N 2 + Fe(CO) 3 , N 2 + Fe(CO) 4 , CO + Fe(CO) 3 (N 2 ), N(C 2 H 5 ) 3 + Fe(CO) 3 , and N(C 2 H 5 ) 3 + Fe(CO) 4 are reported. The magnitudes of these rate constants are compared to rate constants for other reactions of Fe(CO) 3 and Fe(CO) 4 in the gas phase. The aforementioned reactions involving Fe(CO) 4 and Fe(CO) 3 (N 2 ) are anticipated to be formally spin disallowed. Factors that could influence the magnitude of the rate constants in these systems are discussed.

Time-resolved IR spectroscopy has been used to study the oxidative addition of H2 to Fe(CO)4 and its reverse reaction, the reductive elimination of H2 from Fe(CO)4H2, in the gas phase. The rate con...

A study of sodium copper chlorophyllin adsorbed on silver colloids (CuChl/Ag) is conducted using surface-enhanced resonance Raman scattering (SERRS) and visible extinction spectroscopy to examine how the system changes as a function of pH. Initially at basic pH, SERRS signal is not detected even though CuChl is adsorbed onto the silver surface and deprotonated. Upon decreasing the solution pH slightly, colloidal aggregation is induced, evidenced by the broadening of the visible extinction spectra. The larger aggregates possess a surface plasmon that is in resonance with the laser excitation frequency (633-nm) and SERRS signal is detected. As the acid protonates CuChl, the overall negatively-charged surface approaches neutrality which induces more aggregation. Complete protonation of CuChl by pH 4.6 results in colloidal precipitation. However, when aggregation is halted about pH 5, adding NaOH(aq) to the system maintains the extent of aggregation and an intense SERRS signal is detected at basic pH.

The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary beta-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-A structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys142 and Lys20. Analyses of the crystallographic structure also suggest a role for Glu161 in the apurinic/apyrimidinic lyase chemistry. The beta-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes beta and beta-delta elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre- and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.

To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

MutY from Escherichia coli is a DNA mismatch repair enzyme involved in the base excision repair pathway. It is an adenine glycosylase which removes adenine when mispaired with guanine, cytosine or 7,8dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a common DNA oxidative damage lesion and mutant strains of E. coli that lack MutY activity have elevated rates of G:C to T:A tranversions (Nghiem et al., 1988). Trypsin produced an N-terminal domain of residues 1–225, p26, and a C-terminal domain of 226–350, p13 (Manuel et al., 1996). The catalytic activity of the enzyme was found solely in the N-terminal domain. Recent work has determined the crystal structure of the p26 domain; the protein has a helix-hairpin-helix structural motif in common with a number of DNA glycosylases and DNA glycosylase/AP lysases (Guan et al., 1998). The crystal structure suggests that MutY utilizes a nucleotide flipping mechanism, in which the adenine is moved to an extrahelical position within the DNA, into an active site pocket where it is excised. Studies of intact MutY and the N-terminal domain show that the C-terminal domain affects substrate binding and mismatch repair activity. Manuel and Lloyd (1997) found that the largest differences between MutY and p26 in binding of natural substrates involved A:G and in mismatch activity A:C. Recent biochemical data suggest that the C-terminal domain is the principal determinant of 8-oxoG specificity (Noll et al., 1999).