Biomedical subjects

Walter A Baase

Publications and source records attributed to Walter A Baase.

14 recordsLinked to original sources

PubMed2006

Sequential reorganization of beta-sheet topology by insertion of a single strand.

Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%-50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands were inserted into two different loops of the beta-sheet, and the structures were determined. Not surprisingly, one insert "loops out" at its insertion site and forms a new small beta-hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the beta-sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C-terminal end of the same beta-strand. Reasons as to why a non-native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.

Amino Acid Motifs↗

PubMed2006

Guanidinium derivatives bind preferentially and trigger long-distance conformational changes in an engineered T4 lysozyme.

The binding of guanidinium ion has been shown to promote a large-scale translation of a tandemly duplicated helix in an engineered mutant of T4 lysozyme. The guanidinium ion acts as a surrogate for the guanidino group of an arginine side chain. Here we determine whether methyl- and ethylguanidinium provide better mimics. The results show that addition of the hydrophobic moieties to the ligand enhances the binding affinity concomitant with reduction in ligand solubility. Crystallographic analysis confirms that binding of the alternative ligands to the engineered site still drives the large-scale conformational change. Thermal analysis and NMR data show, in comparison to guanidinium, an increase in protein stability and in ligand affinity. This is presumably due to the successive increase in hydrophobicity in going from guanidinium to ethylguanidinium. A fluorescence-based optical method was developed to sense the ligand-triggered helix translation in solution. The results are a first step in the de novo design of a molecular switch that is not related to the normal function of the protein.

Amino Acid Sequence↗

PubMed2005

Low energy CD of RNA hairpin unveils a loop conformation required for lambdaN antitermination activity.

N protein coded by phage lambda is a transcription factor that stimulates the antitermination activity of Escherichia coli RNA polymerase by binding specifically to the nascent RNA transcript at a stemloop structure called boxB. We use a new biophysical technique, involving the monitoring of the low energy circular dichroism spectra of 2-aminopurine residues site-specifically placed in the boxB RNA loop, to investigate this binding interaction. The low energy CD spectra of these 2-aminopurine probes reflect specific asymmetric interactions with adjacent nucleotide bases. Consequently, these CD spectra provide detailed and specific conformational information about the RNA chain at these chromophores that cannot be obtained from changes in the related fluorescence signals of these probes. CD changes were observed on binding the N peptide to boxB RNA that correspond to structural changes that had been previously seen by NMR, thus validating our experimental approach. The low energy CD method was then used to quantify the ordered and disordered states of the free hairpin loop and to show that a significant fraction of the boxB loop assumes a product-like structure in the absence of protein. A boxB derivative with an intact stem and a reduced concentration of ordered loop was identified and used to show that the extent of the reaction between protein and boxB depends on the concentration of structured loop in the RNA reactant population. This result has general implications for the conformational specificity of RNA-protein interactions.

2-Aminopurine↗

PubMed2005

Investigating local conformations of double-stranded DNA by low-energy circular dichroism of pyrrolo-cytosine.

Local base stacking and conformational mobility play a major role in the structure and function of nucleic acids. We have recently shown that the low-energy CD spectrum of 2-aminopurine (2-AP), i.e., the CD spectral region above 300 nm, can be used to monitor conformational changes in polynucleotides at or near mono- and dinucleotide 2-AP residues that replace adenine residues in DNA and RNA. Here, we extend this technique to pyrrolo-cytosine (PC), a fluorescent analogue of cytosine. The low-energy CD spectrum of a PC dinucleotide in dsDNA exhibits an exciton couplet with two bands of opposite sign centered at 350 nm. This signal is characteristic of base stacking between adjacent PC residues in a right helical conformation. Isolated PC nucleotide residues inserted into polynucleotide chains also display chirality that reflects the asymmetric environment of their sequence context. Thus, we show that the low-energy CD spectra of C(PC)A and A(PC)C sequences in dsDNA have opposite signs. It appears that the measurement of the low-energy CD spectra of PC residues will usefully complement 2-AP measurements by serving to characterize the local conformations and dynamics of nucleic acids near C residues and G-C base pairs.

Circular Dichroism↗

PubMed2004

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation.

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.

Alanine↗

PubMed2004

Use of sequence duplication to engineer a ligand-triggered, long-distance molecular switch in T4 lysozyme.

We have designed a molecular switch in a T4 lysozyme construct that controls a large-scale translation of a duplicated helix. As shown by crystal structures of the construct with the switch on and off, the conformational change is triggered by the binding of a ligand (guanidinium ion) to a site that in the wild-type protein was occupied by the guanidino head group of an Arg. In the design template, a duplicated helix is flanked by two loop regions of different stabilities. In the "on" state, the N-terminal loop is weakly structured, whereas the C-terminal loop has a well defined conformation that is stabilized by means of nonbonded interactions with the Arg head group. The truncation of the Arg to Ala destabilizes this loop and switches the protein to the "off" state, in which the duplicated helix is translocated approximately 20 A. Guanidinium binding restores the key interactions, restabilizes the C-terminal loop, and restores the "on" state. Thus, the presence of an external ligand, which is unrelated to the catalytic activity of the enzyme, triggers the inserted helix to translate 20 A away from the binding site. The results illustrate a proposed mechanism for protein evolution in which sequence duplication followed by point mutation can lead to the establishment of new function.

Bacteriophage T4↗

PubMed2004

Low-energy circular dichroism of 2-aminopurine dinucleotide as a probe of local conformation of DNA and RNA.

Circular dichroism is commonly used to investigate the conformations of nucleic acids. However, many biochemical processes implicate conformational changes of particular nucleotide residues within DNA or RNA that cannot be studied by this method, because the CD of these residues is buried in the total signal of the polynucleotide. Here, we report a method to study local conformations of DNA or RNA that is based on the use of the CD of 2-aminopurine (AP) residues as a probe. AP is readily incorporated into DNA in place of adenine and does not significantly alter DNA structure. Unlike adenine, AP is fluorescent and this property has been used for many years to investigate local nucleic acid structure. We show here that the CD spectrum of AP dinucleotide, (AP)(2), exhibits a positive CD band at 326 nm, a spectral region in which nucleic acids (and proteins) do not absorb. Our results show that the bases of (AP)(2) are stacked in a right-handed helical conformation. A low-energy CD band is also observed when this nucleotide dimer is incorporated into double-stranded DNA. Control experiments show that this signal comes from the stacking of adjacent AP residues. We have used this CD signal to provide information about the conformation of the AP dinucleotide at a defined position within single- and double-stranded nucleic acids.

2-Aminopurine↗

PubMed2004

Relocation or duplication of the helix A sequence of T4 lysozyme causes only modest changes in structure but can increase or decrease the rate of folding.

In T4 lysozyme, helix A is located at the amino terminus of the sequence but is associated with the C-terminal domain in the folded structure. To investigate the implications of this arrangement for the folding of the protein, we first created a circularly permuted variant with a new amino terminus at residue 12. In effect, this moves the sequence corresponding to helix A from the N- to the C-terminus of the molecule. The protein crystallized nonisomorphously with the wild type but has a very similar structure, showing that the unit consisting of helix A and the C-terminal domain can be reconstituted from a contiguous polypeptide chain. The protein is less stable than the wild type but folds slightly faster. We then produced a second variant in which the helix A sequence was appended at the C-terminus (as in the first variant), but was also restored at the N-terminus (as in the wild type). This variant has two helix A sequences, one at the N-terminus and the other at the C-terminus, each of which can compete for the same site in the folded protein. The crystal structure shows that it is the N-terminal sequence that folds in a manner similar to that of the wild type, whereas the copy at the C-terminus is forced to loop out. The stability of this protein is much closer to that of the wild type, but its rate of folding is significantly slower. The reduction in rate is attributed to the presence of the two identical sequence segments which compete for a single, mutually exclusive, site.

Bacteriophage T4↗

PubMed2004

Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins.

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

Binding Sites↗

PubMed2003

Repacking the Core of T4 lysozyme by automated design.

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

Automation↗

PubMed2003

Multiple methionine substitutions are tolerated in T4 lysozyme and have coupled effects on folding and stability.

In order to further explore the tolerance of proteins to amino acid substitutions within the interior, a series of core residues was replaced by methionine within the C-terminal domain of T4 lysozyme. By replacing leucine, isoleucine, valine and phenylalanine residues a total of 10 methionines could be introduced, which corresponds to a third of the residues that are buried in this domain. As more methionines are incorporated the protein gradually loses stability. This is attributed in part to a reduction in hydrophobic stabilization, in part to the increased entropic cost of localizing the long, flexible methionine sidechains, and in part to steric clashes. The changes in structure of the mutants relative to the wildtype protein are modest but tend to increase in an additive fashion as more methionines are included. In the most extreme case, namely the 10-methionine mutant, much of the C-terminal domain remains quite similar to wildtype (root-mean-square backbone shifts of 0.56 A), while the F and G helices undergo rotations of approximately 20 degrees and center-of-mass shifts of approximately 1.4 A. For up to six methionine substitutions the changes in stability are additive. Beyond this point, however, the multiple mutants are somewhat more stable than suggested from the sum of their constituents, especially for those including the replacement Val111-->Met. This is interpreted in terms of the larger structural changes associated with this substitution. The substituted sidechains in the mutant structures have somewhat higher crystallographic thermal factors than their counterparts in WT*. Nevertheless, the interiors of the mutant proteins retain a well-defined structure with little suggestion of molten-globule characteristics. Lysozymes in which selenomethionine has been incorporated rather than methionine tend to have increased stability. At the same time they also fold faster. This provides further evidence that, at the rate-limiting step in folding, the structure of the C-terminal domain of T4 lysozyme is similar to that of the fully folded protein.

Amino Acid Substitution↗

PubMed2002

A helix initiation signal in T4 lysozyme identified by polyalanine mutagenesis.

To better understand the relation between sequence and structure, and in an attempt to simplify the protein folding problem, a series of alanine substitutions was introduced into bacteriophage T4 lysozyme. In contrast to previous studies in this system, which were restricted to single alpha-helices, the present analysis included a helix-turn-helix region, a loop-helix region, and two alpha-helices that were well separated in the three-dimensional structure. It was shown previously that T4 lysozyme is very tolerant of alanine substitutions within alpha-helices, especially at solvent-exposed sites. The present study shows that the protein is also tolerant of such substitutions in turn and loop regions, although less than in helices. The results confirm that the structural information in the amino acid sequence is highly redundant. For example, the protein with the sequence 127AAAAAALAAAAWAAA141 folds normally, has melting temperature only 0.8 degrees C lower than wildtype, and has a crystal structure that is also very similar to wildtype. Polyalanine substitutions within turns or loops can, however, lead to differences in structure and in folding. In one example the triple substitution K35A/S36A/P37A caused this region of the molecule to change to a more helical conformation. In a second case the mutant with the sequence 34AAAAALAAAKAALAAA49, which spans a loop-helix region, had a dramatically altered thermal unfolding transition, suggesting that this region may tend to form a single, uninterrupted, helix. Substitution of Ala38 in the above construct with aspartic acid caused the unfolding to be more like wildtype, suggesting that residue 38, which is at a helix-capping position in the wildtype structure, provides an initiation signal that is essential in the polyalanine mutant for the correct formation of alpha-helix 39-50. In a typical protein, the information that codes for the 3D structure is presumably distributed over many amino acids. The present results suggest that in simplified sequences the key folding information may be restricted to a subset of critical residues, and so be more readily accessible to experimental analysis.

Amino Acid Sequence↗

PubMed2002

A model binding site for testing scoring functions in molecular docking.

Prediction of interaction energies between ligands and their receptors remains a major challenge for structure-based inhibitor discovery. Much effort has been devoted to developing scoring schemes that can successfully rank the affinities of a diverse set of possible ligands to a binding site for which the structure is known. To test these scoring functions, well-characterized experimental systems can be very useful. Here, mutation-created binding sites in T4 lysozyme were used to investigate how the quality of atomic charges and solvation energies affects molecular docking. Atomic charges and solvation energies were calculated for 172,118 molecules in the Available Chemicals Directory using a semi-empirical quantum mechanical approach by the program AMSOL. The database was first screened against the apolar cavity site created by the mutation Leu99Ala (L99A). Compared to the electronegativity-based charges that are widely used, the new charges and desolvation energies improved ranking of known apolar ligands, and better distinguished them from more polar isosteres that are not observed to bind. To investigate whether the new charges had predictive value, the non-polar residue Met102, which forms part of the binding site, was changed to the polar residue glutamine. The structure of the resulting Leu99Ala and Met102Gln double mutant of T4 lysozyme (L99A/M102Q) was determined and the docking calculation was repeated for the new site. Seven representative polar molecules that preferentially docked to the polar versus the apolar binding site were tested experimentally. All seven bind to the polar cavity (L99A/M102Q) but do not detectably bind to the apolar cavity (L99A). Five ligand-bound structures of L99A/M102Q were determined by X-ray crystallography. Docking predictions corresponded to the crystallographic results to within 0.4A RMSD. Improved treatment of partial atomic charges and desolvation energies in database docking appears feasible and leads to better distinction of true ligands. Simple model binding sites, such as L99A and its more polar variants, may find broad use in the development and testing of docking algorithms.

Algorithms↗

PubMed2002

A test of proposed rules for helix capping: implications for protein design.

alpha-helices within proteins are often terminated (capped) by distinctive configurations of the polypeptide chain. Two common arrangements are the Schellman motif and the alternative alpha(L) motif. Rose and coworkers developed stereochemical rules to identify the locations of such motifs in proteins of unknown structure based only on their amino acid sequences. To check the effectiveness of these rules, they made specific predictions regarding the structural and thermodynamic consequences of certain mutations in T4 lysozyme. We have constructed these mutants and show here that they have neither the structure nor the stability that was predicted. The results show the complexity of the protein-folding problem. Comparison of known protein structures may show that a characteristic sequence of amino acids (a sequence motif) corresponds to a conserved structural motif. In any particular protein, however, changes in other parts of the sequence may result in a different conformation. The structure is determined by sequence as a whole, not by parts considered in isolation.

Amino Acid Motifs↗