Solution structure of the cold-shock-like protein from Rickettsia rickettsii.

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 ß-strands 3 and 4, the solution structure of the R. rickettsii cold-shock-like protein has the typical Greek-key five-stranded ß-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.

SERRS and visible extinction spectroscopy of copper chlorophyllin on silver colloids as a function of pH.

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.

Reaction intermediates in the catalytic mechanism of Escherichia coli MutY DNA glycosylase.

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.