Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.The biological role of the E colicin-binding immunity proteins Im2, Im7, Im8, and Im9 is to protect the host cell from the toxic endonuclease activity of a coexpressed cognate colicin, by binding to an exosite in the DNase domain, inhibiting access of the substrate DNA to the active site of the colicin by steric hindrance and electrostatic repulsion (1, 2). The small size of the immunity proteins (85-87 amino acid residues), their high level of sequence identity (approximately 60%), the single conserved tryptophan, and their lack of disulfide bonds, prosthetic groups, and cis-prolines in the native state make the immunity protein family ideal for protein folding studies. Despite the high degree of similarity between Im7 and Im9 in both sequence and structure, these proteins fold by mechanisms with different levels of kinetic complexity at neutral pH, suggesting that local sequence variations, rather than topology or global stability, are responsible for the observed folding kinetics of these proteins (3-7). Im7* 1 and Im9* (hexahistidinetagged analogues of Im7 and Im9) contain four helices ( Figure 1A), each of which displays polar side chains on the surface of the protein, while the majority of hydrophobic side chains are buried, forming a well-packed hydrophobic core. Im7* and Im...

A reliable device that produces efficient mixing with a short dead time has enormous utility in the kinetic analysis of biochemical and chemical processes. We have designed two different T mixers that use moderate flow rates (0.2–0.4ml∕s), can monitor reactions up to several milliseconds, and achieve mixing times as low as 20μs. The two mixers are easy to build and dismantle, reliable, and can perform hundreds of experiments without blocking. The first mixer comprises a stainless steel block, containing a microchannel, glued to a quartz cuvette, containing a 200×200μm2 observation channel defining a conventional T mixer. The reactions are monitored by imaging the length of the observation channel onto a charge-coupled device camera. In the second mixer the entire T (200×200μm2 internal cross section) is contained within a 40-mm-long quartz cuvette. We have adopted a novel approach to controlling the entrance channel bore by inserting a stainless steel wire in order to increase the linear speed of the impinging fluids. Using a dye to visualize the flow profile inside the second T mixer, it was shown that in this T geometry segregation of the reactants is observed in the junction between the inlet channels and the observation channel (T junction) and mixing occurs entirely in the observation channel. We thoroughly tested the two mixers through several kinetic reactions using both fluorescence and ultraviolet resonance Raman spectroscopy measurements. We show that both mixers provide efficient mixing with nominal dead times (using 1:10 v∕v dilution), calculated using the quenching of the fluorescence of N-acetyl-L-tryptophanamide by N-bromosuccinimide, of 200±20 and 100±10μs, for each mixer, respectively. However, the ability to monitor within the inlet channels and the entire observation channel of the second mixer shows that this standard approach to estimating the dead time is artifactual, since it relies on assuming a constant flow speed throughout the observation channel, a feature that we show is not adhered to at short distances from the T junction. Using both mixers the refolding of the A state of cytochrome c to the native state was followed by fluorescence and ultraviolet resonance Raman spectroscopy, revealing the ability of these instruments to provide insights into the early stages of protein folding using only milligrams of sample.