The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13 C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins. C ataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer's disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6-8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8-10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mech...

The high specificity of incorporation of nucleotides into DNA by polymerase enzymes is crucial for maintaining fidelity of information transfer in cellular replication. The initial insertion event is the first point at which mutations of the genome are avoided. 1,2 Mismatched pairing at this step occurs on the level of only ∼1 in 10 3 -10 5 insertions, indicating a selectivity of at least 4 kcal/mol. 1 It is clear that polymerases enhance the selectivity of nucleotide choice at the active site relative to the much lower pairing differences observed at the duplex terminus in the absence of enzyme. 2 While many of the kinetic details of replication have been studied in recent years, 3 the precise physical origins of this selectivity enhancement are poorly understood. Mechanisms involving both kinetic and binding selectivity between correct and incorrect nucleotides have been proposed. 3 Base-base hydrogen bonding, base stacking, base pair geometry, and interactions between the enzyme, DNA, and nucleotides have all been invoked as potentially important interactions; however, the relative importance of these different effects remains unclear. Many DNA nucleotide analogs with altered or reduced H-bonding potential have been examined as substrates for polymerases; 4 most of those analogs are quite poor substrates, and result in less discriminate incorporation fidelity than do the natural nucleotides. This general finding has been used as evidence that the number and strength of hydrogen bonds in a given pair determine efficiency and fidelity of DNA synthesis. Indeed, most if not all current models for replication fidelity hold that the specificity of hydrogen bonds formed in the new base pair is a central contributor to the observed selectivity.Here we present evidence, however, that a DNA polymerase can exert high fidelity even when a base pair completely lacks conventional hydrogen bonds. The difluorotoluene nucleoside 1 has recently been constructed as a nonpolar shape mimic for natural thymidine (2). 5,6 Its "base" moiety cannot measurably form paired complexes with adenine derivatives even in chloroform, a solvent in which H-bonded complexes are much more stable than they are in water. 7 When placed within a DNA strand paired opposite adenine, moreover, it actually destabilizes the helix by ∼4-5 kcal relative to thymine at the same position. In addition, 1 shows no inherent pairing selectivity among the four natural bases, also consistent with its nonpolar, nonhydrogen-bonding nature (ref 7 and work in progress). We felt therefore that 1 would serve as a good test for the importance of thymidine's hydrogen bonding groups on fidelity, because 1 lacks the strongly localized charges but retains nearly the exact steric shape of the natural molecule. If, as current models suggest, such polar interactions are important for achieving high fidelity, then 1 would be expected to be very inefficient and highly nonselective as a template for replication.© 1997 American Chemical Society * Author to whom correspondence should...

There is an enormous amount of interest in the structures and formation mechanisms of amyloid fibers. In this Perspective, we review the most common structural motifs of amyloid fibers and discuss how infrared spectroscopy and isotope labeling can be used to identify their structures and aggregation kinetics. We present three specific strategies, site-specific labeling to obtain residue-by-residue structural information, isotope dilution of uniformly labeled proteins for identifying structural folds and protein mixtures, and expressed protein ligation for studying the domain structures of large proteins. For each of these methods, vibrational couplings are the source of the identifying features in the infrared spectrum. Examples are provided using the proteins hIAPP, Aβ, polyglutamine, and γD-crystallin. We focus on FTIR spectroscopy but also describe new observables made possible by 2D IR spectroscopy.

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo ؊ mutant) of Escherichia coli DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (V max ͞K m ) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F-A pair is associated with a brief pause, whereas that beyond A in the inverted A-F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A-F͞F-A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide͞ template shape complementarity may play a more important role than previously believed.