[On chemical studies of the tertiary structure of proteins. II. Analysis of azoglobin, separation and identification of individual bridged peptides].
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Animals↗
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Results for “Protein Structure, Tertiary”
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[Calculation of the tertiary structure of proteins on the basis of an analysis of the matrix contacts between amino acid residues].
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Amino Acids↗
[Search for sites containing functionally important substitutions in series of related or mutant proteins].
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Disintegrins↗
Variable conformation and dynamics of calmodulin complexed with peptides derived from the autoinhibitory domains of target proteins.
Calcium-saturated calmodulin (CaM) can bind and activate many target proteins through the direct association with the respective autoinhibitory domains. The CaM binding sequences within the autoinhibitory domains of these proteins have little sequence homology, and the mechanisms associated with CaM's ability to recognize and productively bind with these variable sequences is unclear. Common structural features of CaM bound to five peptides that are homologous to the autoinhibitory domains of smooth muscle myosin light chain kinase, CaM-dependent protein kinase II alpha, the plasma membrane Ca-ATPase, a MARCKS homolog, and glycogen phosphorylase kinase were assessed using frequency-domain fluorescence spectroscopy. In addition, the structural features of CaM complexed with the peptide melittin was also considered. We observe similar decreases in the average fluorescence lifetime and similar increases in the solvent accessibility of N-(1-pyrenyl)maleimide (PM) bound at Cys27 in calcium binding loop I in the amino terminal domain of CaM upon association with all six target peptides. Likewise, using fluorescence resonance energy transfer to measure the spatial separation between the opposing globular domains in CaM, we observe a similar spatial separation between the opposing globular domains of CaM bound to all six peptides. This indicates that CaM undergoes comparable structural changes upon association with all six target peptides. However, there are significant differences in the observed lifetime, solvent accessibility, correlation time associated with the segmented rotational motion of PM-CaM, and in the spatial separation between the opposing globular domains in CaM upon association with the individual target peptides, which indicates that CaM adopts a different tertiary structure that is dependent on the structural features of the bound target peptide. The correlation times associated with the overall hydrodynamic properties of CaM complexed with all six peptides are nearly identical (phi 2 approximately 10.6 +/- 0.4 ns) and are consistent with the known dimensions of CaM complexed to a peptide homologous to the CaM binding sequence of CaM-dependent protein kinase II alpha. Therefore, while these results are consistent with a common binding mechanism between CaM and all six target peptides, they indicate that the binding domains of CaM adopt different tertiary structures that allow them to bind with the variable sequences found in the autoinhibitory domains of target proteins with high affinity.
Amino Acid Sequence↗
A designed branched three-helix bundle protein dimer.
The ultimate goals of de novo protein design are the construction of novel tertiary structures and functions. Here is presented the design and synthesis of a uniquely branched three-helix bundle that folds into a well-folded dimeric protein. The branching of this protein was performed by the method of native chemical ligation, which provides a chemoselective and stable amide bond between the unprotected fragments. This ligation strategy was possible by the presented facile preparation of a peptide (43 amino acids) with a specific side chain thioester, which is synthesized by general Fmoc solid phase peptide synthesis. From the presented structural analysis, it is seen that the folded protein is present as a stable and highly helical dimer, thus forming a six-helix bundle. This unique tertiary structure, composed of a dimer of three individual alpha-helices branched together, offers different possibilities for protein engineering, such as metal and cofactor binding sites, as well as for the construction of novel functions.
Amino Acid Sequence↗
Effect of a specific hydrogen bond (N138ND2-Q106O) on conformational integrity, stability, and activity of staphylococcal nuclease.
There are two hydrogen bonding interactions (N138ND2-Q106O and Y54OH-S141OG) between the C-terminal region and the main body of staphylococcal nuclease (SNase). To examine the role of these hydrogen bonds, SNase(141) and its three mutants, SNase(141)N138D, SNase(141)S141A, and SNase(141)N138D/S141A, were created. The N138D mutation has the N138ND2-Q106O interaction deleted and the S141A mutation has the Y54OH-S141OG and S141OG-N138O interactions deleted. The conformational features, stability, and activity of the proteins have been compared by using circular dichroism, intrinsic and ANS-binding fluorescence, GdnHCl-induced denaturation, and activity assay. The results clearly show that the N138D mutation significantly alters the secondary and tertiary structures of the protein, producing a partially unfolding state; in contrast, the S141A mutation has no such effect on structure. These results strongly suggest that the specific hydrogen bond, N138ND2-Q106O, plays an important role in maintaining the conformational integrity and stability of the nuclease.
Enzyme Activation↗
Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.
Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.
Alkyl and Aryl Transferases↗
Rigid domains in proteins: an algorithmic approach to their identification.
A rigid domain, defined here as a tertiary structure common to two or more different protein conformations, can be identified numerically from atomic coordinates by finding sets of residues, one in each conformation, such that the distance between any two residues within the set belonging to one conformation is the same as the distance between the two structurally equivalent residues within the set belonging to any other conformation. The distance between two residues is taken to be the distance between their respective alpha carbon atoms. With the methods of this paper we have found in the deoxy and oxy conformations of the human hemoglobin alpha 1 beta 1 dimer a rigid domain closely related to that previously identified by Baldwin and Chothia (J. Mol. Biol. 129: 175-220, 1979). We provide two algorithms, both using the difference-distance matrix, with which to search for rigid domains directly from atomic coordinates. The first finds all rigid domains in a protein but has storage and processing demands that become prohibitively large with increasing protein size. The second, although not necessarily finding every rigid domain, is computationally tractable for proteins of any size. Because of its efficiency we are able to search protein conformations recursively for groups of non-intersecting domains. Different protein conformations, when aligned by superimposing their respective domain structures, can be examined for structural differences in regions complementing a rigid domain.
Algorithms↗
Identifying the tertiary fold of small proteins with different topologies from sequence and secondary structure using the genetic algorithm and extended criteria specific for strand regions.
Grid-free protein folding simulations based on sequence and secondary structure knowledge (using mostly experimentally determined secondary structure information but also analysing results from secondary structure predictions) were investigated using the genetic algorithm, a backbone representation, and standard dihedral angular conformations. Optimal structures are selected according to basic protein building principles. Having previously applied this approach to proteins with helical topology, we have now developed additional criteria and weights for beta-strand-containing proteins, validated them on four small beta-strand-rich proteins with different topologies, and tested the general performance of the method on many further examples from known protein structures with mixed secondary structural type and less than 100 amino acid residues. Topology predictions close to the observed experimental structures were obtained in four test cases together with fitness values that correlated with the similarity of the predicted topology to the observed structures. Root-mean-square deviation values of C alpha atoms in the superposed predicted and observed structures, the latter of which had different topologies, were between 4.5 and 5.5 A(2.9 to 5.1 A without loops). Including 15 further protein examples with unique folds, root-mean-square deviation values ranged between 1.8 and 6.9 A with loop regions and averaged 5.3 A and 4.3 A, including and excluding loop regions, respectively.
Algorithms↗
Comparison of atomic solvation parametric sets: applicability and limitations in protein folding and binding.
Atomic solvation parameters (ASP) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein-ligand complexes. They are also included in several molecular mechanics computer programs. In this work, a total of eight atomic solvation parametric sets has been employed to calculate the solvation contribution to the free energy of folding delta Gs for 17 proteins. A linear correlation between delta Gs and the number of residues in each protein was found for each ASP set. The calculations also revealed a great variety in the absolute value and in the sign of delta Gs values such that certain ASP sets predicted the unfolded state to be more stable than the folded, whereas others yield precisely the opposite. Further, the solvation contribution to the free energy of association of helix pairs and to the disassociation of loops (connection between secondary structural elements in proteins) from the protein tertiary structures were computed for each of the eight ASP sets and discrepancies were evident among them.
Chemical Phenomena↗
Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage.
This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.
Animals↗
Initiation of DNA replication by the Escherichia coli dnaG protein: evidence that tertiary structure is involved.
The dnaG protein of Escherichia coli initiates DNA replication by synthesizing primer oligonucleotides for elongation by DNA polymerae. The experiments reported here probe the nature of the nucleic acid element recognized by the dnaG protein. Three well-separated groups of nucleotides within the negative-strand origin of the single-stranded phage phi K are protected by the dnaG protein against nuclease digestion. DNA as far as 115 bases from the start site of primer synthesis is involved in binding of the dnaG protein to the replication origin. One molecule of dnaG protein could protect all of these nucleotides if the DNA were folded into a higher-order tertiary structure. Protection of the phi K origin by dnaG protein requires DNA binding protein, and it does not occur if the group of protected nucleotides most distant from the start site is removed from the template. There is no binding of dnaG protein to the complementary strand of the phi K origin-region DNA. The observed protection of the positive strand is due to a functional nucleic acid-protein complex.
Bacterial Proteins↗
Early events in protein folding.
Recent advances have significantly increased the time and spectroscopic resolution of protein folding experiments. We can now study the timescale and nature of polypeptide collapse, and how this correlates with secondary and tertiary structure formation. Studies on ultrafast folding proteins and peptides provide experimental benchmarks on a timescale that overlaps directly with that of molecular dynamics simulations. This makes possible direct tests of both simulations and current models of protein folding.
Models, Molecular↗
Structural comparison of lactose permease and the glycerol-3-phosphate antiporter: members of the major facilitator superfamily.
Structural knowledge of the major facilitator superfamily has dramatically increased during the past year with the emergence of the structures of three members of this family of transporters. All three structures reveal 12 transmembrane helices forming two distinct domains, and could imply that members of this superfamily have preserved both secondary and tertiary structure elements during evolution.
Binding Sites↗
A common sequence-associated physicochemical feature for proteins of beta-trefoil family.
Different amino acid sequences can fold into similar tertiary structures but the reasons for it are not very clear. It has been suggested in the literature that these sequences may have some common features associated with them but the exact nature of such shared properties remains largely unknown. We studied a representative sample of proteins from the beta-trefoil family and observed that their amino acid sequences, despite being considerably divergent from each other, can be accounted for by matching to a repetition of three physicochemically similar segments. This observation in turn is consistent with the three-fold pseudo-symmetry in tertiary structures of these proteins.
Amino Acid Sequence↗
Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.
Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.
Allosteric Site↗
Separation techniques for high-molecular-mass proteins.
Many high-molecular-mass (HMM) proteins (MW>100 kDa) are known to be involved in cytoskeleton, defence and immunity, transcription and translation in higher eukaryotic organisms. Even in the post-genomic era, purification of HMM protein is the first important step to analyze protein composition in a tissue or a cell (proteomics), to determine protein tertiary structure (structural biology), and to investigate protein function (functional genomics). To separate a HMM protein from a protein mixture, ions, chaotropes (urea and thiourea), detergents and protease inhibitors in extraction media and buffer solutions either for liquid chromatography or for gel electrophoresis should be carefully chosen, since HMM proteins tend to be aggregates under denatured condition and their long polypeptide chains are easily attacked by intrinsic proteases during separation procedure. Among many liquid chromatography techniques, affinity chromatography either with sequence-specific DNA for transcription factor, or with monoclonal antibody specific for myosin heavy chain has been used for preparative isolation of the respective HMM proteins. Though SDS-PAGE could analyze the size and the quantity of megadalton proteins, the resolution of HMM proteins is relatively poor. A newly developed pulse SDS-PAGE would be able to raise the resolution of HMM proteins compared with the conventional SDS-PAGE. The 2-DE method is not particularly suitable in analyzing HMM proteins larger than 200 kDa. However, a 2-DE method that uses an agarose IEF gel in the first dimension (agarose 2-DE) has been shown to produce significant improvements in 2-DE separation of HMM proteins larger than 150 kDa and up to 500 kDa.
Chromatography, Liquid↗
Mapping of heme-binding domains in soluble guanylyl cyclase beta1 subunit.
Soluble guanylyl cyclase (sGC) is activated upon the interaction of NO with heme in the sGC beta1 subunit. To identify the domains contributing to heme-binding, we constructed a series of deletion mutants of the beta1 subunit, and evaluated their heme-binding capability. Deletion mutants consisting of residues 1-120 [beta1(1-120)] and 80-385 [beta1(80-385)] were the shortest mutants exhibiting heme binding among the C-terminal and N-terminal-truncated mutants, respectively. The region common to both beta1(1-120) and beta1(80-385), i.e., residues 80-120, is therefore essential for heme binding, although the residues 341-385 play an auxiliary role in heme binding. Two deletion mutants, beta1(80-195) and beta1(60-195), which include only the essential region, exhibited strong heme binding and spectral properties similar to those of the nitrosyl complex of native sGC. Thus, these heme-binding core proteins may serve as model proteins for future studies on the tertiary structure of the nitrosyl complex of sGC.
Animals↗