Jian Xu

The cyclic AMP response element-binding protein (CREB) family can regulate biological functions of various types of cells by forming homo- or heterodimers to bind the target DNA sequences; it plays an essential role in individual neuronal function and entire neuronal circuits. One attractive activity of the CREB family is regulating the transcription of apoptosis-suppressor gene bcl-2. Cyclic AMP response element modulator-1 (CREM-1) is one member of the family with limited acquaintance. To investigate whether CREM-1 is involved in central nervous system injury and repair, we performed an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of CREM-1 in ipsilateral peritrauma cortex. Immunofluorescent labeling indicated that CREM-1 was localized mainly in the nuclei of neurons; co-localization of CREM-1 and active-caspase-3 in the ipsilateral cortex suggested that CREM-1 might participate in neuronal apoptosis. To further investigate the function of CREM-1, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of CREM-1 with p-CREB on PC12 cells by Western blot, immunofluorescent labeling, and co-immunoprecipitation. The result implied that the association of CREM-1 with p-CREB was enhanced in apoptotic cells. Additionally, knocking CREM-1 down with siRNA demonstrated the probable pro-apoptotic role played by CREM-1 in neuronal apoptosis. Together with our data, we hypothesized that CREM-1 might play an important role in regulating neuronal death after TBI by interacting with CREB.

Experiments were set up to test how the crystallization drop size affects the crystallization process; in the test cases studied, increasing the drop size led to increasing numbers of crystals. Other data produced from a high-throughput automation-system run were analyzed in order to gauge the effect of replication on the success of crystallization screening. With over 40-fold multiplicity, lysozyme was found to crystallize in over half of the conditions in a standard 96-condition screen. However, despite the fact that industry-standard lysozyme was used in our tests, it was rare that we obtained crystals reproducibly; this suggests that replication whilst screening might improve the success rate of macromolecular crystallization.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se‐Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X‐ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence‐structure relationships between the SGX and PDB structures were investigated using PDB‐BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Proteins 2005.

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent‐binding sites. Others were intended to displace solvent from preexisting sites. In one case Val‐149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild‐type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr‐152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7Å, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent‐binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen‐bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen‐bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.

The crystal structure of an efficient Diels-Alder antibody catalyst at 1.9 angstrom resolution reveals almost perfect shape complementarity with its transition state analog. Comparison with highly related progesterone and Diels-Alderase antibodies that arose from the same primordial germ line template shows the relatively subtle mutational steps that were able to evolve both structural complementarity and catalytic efficiency.

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non‐polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the “large‐to‐small” substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts ≤ 0.3 Å); in other cases mainchain atoms moved up to 2 Å. In the case of Ile 29 → Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 → Ala (V149A) and Met 6 → Ala (M6A), however, are exceptions and have, respectively, one and two well‐ordered water molecules within the cavity. The Val 149 → Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild‐type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 → Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., − 2.1 kcal/mol for all Leu → Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol ⁻¹ Å ⁻³ , Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu → Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255 :178‐183).

In order to determine the thermodynamic cost of introducing a polar group within the core of a protein, a series of nine Ala-->Ser and 3 Val-->Thr substitutions was constructed in T4 lysozyme. The sites were all within alpha-helices but ranged from fully solvent-exposed to totally buried. The range of destabilization incurred by the Ala-->Ser substitutions was found to be very similar to that for the Val-->Thr replacements. For the solvent-exposed and partly exposed sites the destabilization was modest (approximately less than 0.5 kcal/mol). For the completely buried sites the destabilization was larger, but variable (approximately 1-3 kcal/mol). Crystal structure determinations showed that the Ala-->Ser mutant structures were, in general, very similar to their wild-type counterparts, even though the replacements introduce a hydroxyl group. This is in part because the introduced serines are all within alpha-helices and at congested sites can avoid steric clashes with surrounding atoms by making a hydrogen bond to a backbone carbonyl oxygen in the preceding turn of the helix. The three substituted threonine side chains essentially superimpose on their valine counterparts but display somewhat larger conformational adjustments. The results illustrate how a protein structure will adapt in different ways to avoid the presence of an unsatisfied hydrogen bond donor or acceptor. In the most extreme case, Val 149-->Thr, which is also the most destabilizing variant (delta delta G = 2.8 kcal/mol), a water molecule is incorporated in the mutant structure in order to provide a hydrogen-bonding partner. The results are consistent with the view that many hydrogen bonds within proteins contribute only marginally to stability but that noncharged polar groups that lack a hydrogen-bonding partner are very destabilizing (delta delta G approximately greater than 3 kcal/mol). Supportive of other studies, the alpha-helix propensity of alanine is seen to be higher than that of serine (delta delta G = 0.46 +/- 0.04 kcal/mol), while threonine and valine are similar in alpha-helix propensity.