Paul R. Carey

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

Principally through the efforts of crystallographers, we are being presented with an ever expanding atomic view of the biological world. Although this brings into focus many questions regarding the mysteries of function, techniques are needed that facilitate the transition in our understanding from structure to function. Raman spectroscopy is one of these; because the Raman effect involves an intimate interplay between atomic positions, electron distribution, and intermolecular forces, it sits at the bridgehead between structure and function. Thus, the Raman technique can answer questions that lie at the heart of issues such as ligand macromolecule recognition and enzymatic catalysis. Raman spectroscopy involves analyzing the scattered photons from a laser beam focused into the sample solution (1). The inelastic scattered photons (the Raman spectrum) provide information on molecular vibrations that, in turn, yield data on molecular conformation and environment. At its most effective, Raman spectroscopy can provide exquisite detail from an important site in a much larger macromolecular complex. Although Raman was first applied to the definition of biological molecules in the 1930s (2), the giant has remained drowsy due the difficulties both in obtaining high quality data and in interpreting those data. Considerable advances have been made in these areas in the past few years, and the giant is stirring! A major goal of this review is to provide biochemists with enough information to determine whether the Raman technique could provide structural insights into their systems. The specific issues addressed are which type of systems are amenable to study and what information could be obtained. Practically, present-day sample requirements are for 20 ml of clear solution, where the target molecule is in the 100–300 mM range. Because the number of vibrational modes of a molecule is 3n 2 6, where n is the number of atoms, the complete Raman spectrum of a macromolecule is exceedingly complex. Thus, Raman is most suited to systems where it is possible to focus upon a small region of interest, e.g. a ligandreceptor or enzyme-substrate binding site. Historically, this condition was achieved by using resonance Raman spectroscopy (1) to obtain the intensity-enhanced spectra from chromophores at specific sites in macromolecules. Recent technical advances mean that similar information can now be gleaned from non-chromophoric systems, markedly broadening the application of the technique. The information obtained can be very detailed, exceeding the level of resolution found in x-ray or NMR analyses (3–5). In addition to providing structural data, the Raman spectrum can also reveal changes in the distribution of electrons in a bound ligand and details of active site-ligand interactions, such as hydrogen bonding strengths. Raman spectroscopy is beginning to fulfill its potential to contribute to structural biology because the three roadblocks that impeded its application to biological systems have been all but removed. These were low sensitivity, interference from fluorescence background, and problems with data interpretation. Sensitivity has increased several orders of magnitude with the corresponding decrease in concentration requirements because of advances in optical filters and photon detectors (6). Fluorescence interference is now minimized by using deep-red excitation in the 650–800 nm range, made possible by the advent of photon detectors with high efficiency in this region (7). Problems with interpreting Raman spectra have receded with the availability of “friendly” software packages (8) and ever increasing computational power that enable us to calculate, ab initio, the Raman spectra of midsized molecules (of the size of many ligands or co-factors found at biological sites). Interpretation is further strengthened by a comparison of the calculated and experimental shifts in Raman peak positions when a molecule is substituted with stable isotopes. Recently, this approach has been used to characterize hydrogen bonding in a complex of adenosine deaminase with a transition state analogue (9) and to discriminate between different protonation states of dihydrofolate binding to dihydrofolate reductase (10).

Insulin Assembly Damps Conformational Fluctuations: Raman Analysis of Amide I Linewidths in Native States and Fibrils

The crystal structure of insulin has been investigated in a variety of dimeric and hexameric assemblies. Interest in dynamics has been stimulated by conformational variability among crystal forms and evidence suggesting that the functional monomer undergoes a conformational change on receptor binding. Here, we employ Raman spectroscopy and Raman microscopy to investigate well-defined oligomeric species: monomeric and dimeric analogs in solution, native T(6) and R(6) hexamers in solution and corresponding polycrystalline samples. Remarkably, linewidths of Raman bands associated with the polypeptide backbone (amide I) exhibit progressive narrowing with successive self-assembly. Whereas dimerization damps fluctuations at an intermolecular beta-sheet, deconvolution of the amide I band indicates that formation of hexamers stabilizes both helical and non-helical elements. Although the structure of a monomer in solution resembles a crystallographic protomer, its encagement in a native assembly damps main-chain fluctuations. Further narrowing of a beta-sheet-specific amide I band is observed on reorganization of insulin in a cross-beta fibril. Enhanced flexibility of the native insulin monomer is in accord with molecular dynamics simulations. Such conformational fluctuations may initiate formation of an amyloidogenic nucleus and enable induced fit on receptor binding.

Proinsulin Is Refractory to Protein Fibrillation TOPOLOGICAL PROTECTION OF A PRECURSOR PROTEIN FROM CROSS-β ASSEMBLY

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-β assembly. Protection from fibrillation in β cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific β-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-β assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect β cells from toxic protein misfolding in the endoplasmic reticulum.

Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit.

Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO2− from methylmalonyl‐CoA to pyruvate to yield propionyl‐CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2‐ketobutyrate. The structure reveals a dimer of β8α8 barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the products carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S‐based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites.

Comparing protein–ligand interactions in solution and single crystals by Raman spectroscopy

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix (“in”) or essentially solvent-exposed (“out”). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavins isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core

Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO2− from methylmalonyl‐CoA to pyruvate, yielding propionyl‐CoA and oxaloacetate. The 1.9 Å resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl‐CoA. Overall, the structure reveals two stacked trimers related by 2‐fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl‐CoA is stabilized by interaction with the N‐termini of two α‐helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl‐CoA carboxylase, whose β‐subunit has 50% sequence identity with 12S. A homology model of the propionyl‐CoA carboxylase β‐subunit, based on this 12S crystal structure, provides new insight into the propionyl‐CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.

Proteins can convert to β-sheet in single crystals

Raman microscopy was used to follow conformational changes in single protein crystals. Crystals of native insulin and of the 5S and 12S subunits of the enzyme transcarboxylase showed a mixture of Raman marker bands signifying α‐helix, β‐sheet and nonordered secondary structure. However, by reducing the S–S bonds in the insulin crystal, or by lowering the pH for the 5S crystal, or by soaking substrates into the 12S crystal, the secondary structure in each crystal became predominantly β‐sheet. The β‐form crystals could be dissolved only with difficulty and yielded high–molecular weight protein aggregates, indicating that the β‐sheet formation involves intermolecular contacts. Although their morphology appeared unchanged, the crystals no longer diffracted X‐rays. Using crystals that had not been exposed to laser light, the dye thioflavin T formed highly fluorescent complexes with the “β‐transformed” crystals but not with the native crystals.

Detection of Innersphere Interactions between Magnesium Hydrate and the Phosphate Backbone of the HDV Ribozyme Using Raman Crystallography

A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm-1, and the intensity of the PO2- symmetric stretch, near 1100 cm-1, perturbed by magnesium binding, demonstrating direct observation of -PO2-...Mg2+(H2O)x innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and H218O that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm-1 PO2- feature seen upon magnesium hydrate binding indicates that there are approximately 5 innersphere Mg2+...-O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.

The glmS Ribozyme Tunes the Catalytically Critical pKa of Its Coenzyme Glucosamine-6-phosphate

The glmS ribozyme riboswitch is the first known natural catalytic RNA that employs a small-molecule cofactor. Binding of glucosamine-6-phosphate (GlcN6P) uncovers the latent self-cleavage activity of the RNA, which adopts a catalytically competent conformation that is nonetheless inactive in the absence of GlcN6P. Structural and analogue studies suggest that the amine of GlcN6P functions as a general acid-base catalyst, while its phosphate is important for binding affinity. However, the solution pK(a) of the amine is 8.06 ± 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK(a)s of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK(a) of the amine of GlcN6P to 7.26 ± 0.09 and raises the pK(a) of its phosphate to 6.35 ± 0.09. Remarkably, the pK(a)s of these two functional groups are unchanged from their values for free GlcN6P (8.06 ± 0.05 and 5.98 ± 0.05, respectively) when GlcN6P binds to the catalytically inactive but structurally unperturbed G40A mutant of the ribozyme, thus implicating the ribozyme active site guanine in pK(a) tuning. This is the first demonstration that a ribozyme can tune the pK(a) of a small-molecule ligand. Moreover, the anionic glmS ribozyme in effect stabilizes the neutral amine of GlcN6P by lowering its pK(a). This is unprecedented and illustrates the chemical sophistication of ribozyme active sites.

Inhibition of OXA-1 β-Lactamase by Penems

ABSTRACT The partnering of a β-lactam with a β-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to β-lactam antibiotics mediated by serine β-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D β-lactamase that is resistant to inactivation by tazobactam. The Ki of each penem inhibitor for OXA-1 was in the nM range (Ki of penem 1, 45 ± 8 nM; Ki of penem 2, 12 ± 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 β-lactamase were 0.13 ± 0.01 s−1 and 0.11 ± 0.01 s−1, respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in ≤900 s and the recovery of OXA-1 β-lactamase activity was not detected at 24 h. Covalent adducts of penems 1 and 2 (changes in molecular masses, +306 ± 3 and +321 ± 3 Da, respectively) were identified by electrospray ionization mass spectrometry (ESI-MS). After tryptic digestion of OXA-1 inactivated by penems 1 and 2, ESI-MS and matrix-assisted laser desorption ionization-time-of-flight MS identified the adducts of 306 ± 3 and 321 ± 3 Da attached to the peptide containing the active-site Ser67. The base hydrolysis of penem 2, monitored by serial 1H nuclear magnetic resonance analysis, suggested that penem 2 formed a linear imine species that underwent 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative. Susceptibility testing demonstrated that the penem inhibitors at 4 mg/liter effectively restored susceptibility to piperacillin. Penem β-lactamase inhibitors which demonstrate high affinities and which form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine β-lactamases present in gram-negative bacteria.