• Conserved folding of proteins that share a similar structure motif

The OB-fold (oligonucleotide/oligosaccharide-binding fold) occurs in more than 80 unrelated proteins. It consists of a 5-stranded beta barrel formed from two- and three-stranded beta-sheets. We have studied the folding of three OB-fold proteins that share no detectable sequence homology: SN (staphylococcal nuclease), LysN (anticodon binding domain of Lys-tRNA synthetase) and CspA (Cold shock protein A). Mutagenesis and hydrogen exchange experiments show that the structural motif conserved between the three proteins is more resistant to unfolding than structural elements that are not conserved between the proteins. This is shown in the figure on the right where the site-specific stabilities to unfolding events that expose amide hydrogens to exchange are mapped on the structures (blue is more stable, green is less stable, white is undetermined). In fact, misfolding of the three proteins also appears to be conserved. The first three strands of beta-sheet show the highest propensity for structure under denaturing conditions, and the acid denatured forms of all three proteins aggregate through the mispairing of this partially formed unfulfilled structure. We are currently exploring the structural determinants of conserved motifs in the OB-fold.

Alexandrescu, A. T., Gittis, A., Abeygunawardana, C., & Shortle, D. (1995) “NMR structure of a stable "OB-fold" sub-domain isolated from staphylococcal nuclease”. J. Mol. Biol. 250, 134-143.

Alexandrescu, A.T., Jaravine, V.A., Dames, S.A. & Lamour, F.P. (1999) "NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: The most conserved elements of structure are the most stable to unfolding". J. Mol. Biol. 289, 1041-1054.

Jaravine, V.A., Rathgeb-Szabo, K., & Alexandrescu, A.T. (2000) "Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide". Protein Science 9, 290-301.

Alexandrescu, A.T., & Rathgeb-Szabo, K. (1999) "An NMR investigation of solution aggregation reactions preceding the misassembly of acid denatured cold shock protein A into fibrils". J. Mol. Biol. 291, 1191-1206.

Alexandrescu, A.T., Jaravine, V.A., & Lamour, F.P. (2000) "NMR evidence for progressive stabilization of native-like structure upon aggregation of acid denatured LysN". J. Mol. Biol. 295, 239-255.

Watson, E., Matousek, W.M., Irimies, E.L. & Alexandrescu, A.T. (2007) “Partially folded states of staphylococcal nuclease highlight the conserved structural hierarchy of OB-fold proteins”, Biochemistry, in press.

• Developed a method to study hydrogen exchange in amyloid fibrils

Amyloid fibrils are protein deposits involved in a number of neurodegenerative diseases such as Alzheimer's. Once thought by experts to be inert and impossible to study by solution NMR, we wanted to investigate the flexibility of fibrils and their interactions with solvent. We developed a method to look at exchange of amide hydrogens from the fibrils with solvent. Hydrogens exposed to the surface should exchange easily, those caught up in structure will be protected. In the method, amyloid fibrils are exposed to D2O, and exchange is quenched by flash freezing. The fibrils are solubilized and dissociated in the aprotic solvent DMSO, which enables the exchange history of the fibrils to be read out indirectly from the denatured state of the protein. This method has recently come into wide use; the publication from our lab preceded other reports by one year. In our original paper we proposed that exchange from fibrils of the E.coli protein CspA occurs predominantly through dissociation of protein from the fibrils. This hypothesis was initially met with much resistance, but similar mechanisms have recently been hailed "molecular recycling".

Alexandrescu, A.T. (2001) "An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by the protein CspA". Pac. Symp. Biocomput. 6, 67-78.

• Formation of individual hydrogen bonds during folding of an alpha-helix

A technique developed by Stefan Grzesiek to detect scalar coupling mediated through hydrogen bonds was used to follow alpha-helix formation in the S-peptide of ribonuclease A. The results show that hydrogen bonds are not uniformly populated as alpha-helical structure forms. The hydrogen-bonds follow a stability gradient that decreases from the center to the ends of the helix. The results are roughly consistent with predictions from the Lifson-Roig theory of coil-helix transitions.

Jaravine, V.A., Alexandrescu A. T., & Grzesiek, S. (2001) "Observation of the closing of individual hydrogen bonds during TFE-induced helix formation in a peptide". Protein Science 10, 943-950.

• NMR structure of a homotrimeric coiled coil.

We determined the first NMR structure of a homotrimer. Homooligomers pose a particular problem for NMR because the monomer chains are magnetically equivalent. Additional approaches are thus needed to distinguish NOEs within a chain from those between chains. To solve the NMR structure of the coiled coil trimer from matrilin-1 we first obtained a highly defined structure for the monomers. We then used a self-consistent strategy to assign NOEs between chains, which were identified from isotope-filtered NOE experiments.

Dames, S.A., Wiltscheck, R., Kammerer, R.A., Engel, J., & Alexandrescu, A.T. (1998) "NMR structure of a parallel homotrimeric coiled coil". Nature Struct. Biol. 5, 687-691.

• Residual dipolar couplings in denatured proteins

We showed how dipolar couplings can arise from localized anisotropic structure in denatured proteins, that dipolar couplings are biased towards the most anisotropic structures in the denatured state ensemble, and examined how dynamics and changes in the distributions of conformers in the denatured state ensemble affect couplings.

Alexandrescu, A.T., & Kammerer, R.A. (2003) “Structure and disorder in the ribonuclease S-peptide probed by NMR residual dipolar couplings”. Protein Sci. 12, 2132-2140.

Sallum, C.O., Martel, D.M., Fournier, R.S., Matousek, W.M & Alexandrescu, A.T. (2005) “Sensitivity of NMR residual dipolar couplings to perturbations in folded and unfolded staphylococcal nuclease”. Biochemistry 44, 6392-6403.

• Dynamics in denatured and partially folded states

15N relaxation measurements have been used to investigate dynamics in a number of equilibrium folding intermediates. The figure shows four forms of the enzyme staphylococcal nuclease with different levels of cooperatively stabilized structure. SN-T is the nuclease in a ternary complex with Ca2+ and the inhibitor pdTp, which stabilize the protein. SN is the wild type protein. SN-OB is a folded fragment of the nuclease that is missing 1/3 of the chain from the C-terminus (see below). D131D is a highly denatured fragment of nuclease with near-random-coil NMR spectra. Blue colors indicate low order parameters (related to the amplitude of motion) and rigid structure, red colors indicate large order parameters and flexibility. As the stability of the native state stability decreases, the main-chain is subject to increasingly larger amplitude motions and the dynamic properties of the chain become more heterogeneous. These results paint a picture of unfolding as a fragmentation (or shattering) of the native state structure, and of folding as an accretion of structure in which the mobility of the main chain is frozen-out and initially separate structural elements become increasingly interdependent in order to achieve the maximum cooperativity and stability of the native state.

Alexandrescu, A.T., Jahnke, W., Wiltscheck, R., & Blommers, M.J.J (1996) "Accretion of structure in staphylococcal nuclease: An 15N NMR relaxation study". J. Mol. Biol. 260, 570-587.

• NMR structure of a sub-domain from a cooperatively folded protein.

Staphylococcal nuclease folds with the typical all-or none cooperativity of a single domain protein. In the presence of the two rare global suppressor mutations V66L and G88V (analogous mutations occur for thermostable nucleases found in nature) a 1-103 fragment of nuclease remains folded in the absence of the last 1/3 (55 residues) of the sequence (purple segment). The portion of the structure that remains folded corresponds to the conserved OB-fold motif found in a variety of protein structures. The C-terminal 1/3 of the sequence is certainly not dispensable since it contains part of the enzyme's active site. The fragment has a 1000-fold lower nuclease activity than the wild type, but still classifies as an enzyme (reaction rate is 10^12 fold higher with the fragment). The fact that part of the polypeptide chain of this single-domain protein can be deleted challenges two-state models of protein folding. It also suggests mechanisms by which protein structures can evolve by assimilating pre-existing structural motifs; sacrificing the independence of sub-domains in order to achieve the maximum cooperativity and stability of the new integrated structure.

Alexandrescu, A. T., Gittis, A., Abeygunawardana, C., & Shortle, D. (1995) “NMR structure of a stable "OB-fold" sub-domain isolated from staphylococcal nuclease”. J. Mol. Biol. 250, 134-143.