Showing papers on "Structural biology published in 1996"

Roles of electrostatic interaction in proteins.

Abstract: More than 60 years after the analyses by Linderstrom-Lang and Kirkwood of their hypothetical 'protein' structures, we have now a plethora of experimental evidence and computational estimates of the electrostatic forces in proteins, with very many protein 3D structures at atomic resolution. In the mean time, there were in the beginning, many arguments and suggestions about the roles of electrostatics, mainly from empirical findings and tendencies. A few experimental results indicated that the electrostatic contribution is of the order of several kcal/mol, which was theoretically difficult to reproduce correctly, because a large opposing reaction field should be subtracted from a large, direct Coulombic field. Although the importance of the reaction field was recognized even 70 years ago, appropriate applications to protein molecules were made only in this decade, with the development of numerical computation. Now, an electrostatic molecular surface is one of the most popular pictures in journals of structural biology, indicating that the electrostatic force is one of the important components contributing to molecular recognition, which is a major focus of current biology and biochemistry. The development of NMR techniques has made it possible to observe the individual ionizations of ionizable groups in a protein, in addition to the determination of the 3D structure. Since it does not require any additional probe, each charge state can report the very local and heterogeneous electrostatic potentials working in the protein, without disturbing the original field. From the pKa values, the contributions of electrostatic interactions, ion pairs, charge-dipole interactions, and hydrogen bonds to protein stability have been correctly evaluated. Protein engineering also provides much more information than that obtainable from the native proteins, as the residues concerned can now be easily substituted with other amino acid residues having electrostatically different characteristics. Those experimental results have revealed smaller contributions than previously expected, probably because we underestimated the reaction field effects. Especially, a single ion pair stabilizes a protein only slightly, although a cooperative salt-bridge network can contribute significantly to protein stability. Marginal stabilities of proteins arise from small difference between many factors with driving and opposing forces. In spite of the small contribution of each single electrostatic interaction to the protein stability, the sum of their actions works to maintain the specific 3D structure of the protein. The 'negative' roles of electrostatics, which might destabilize protein conformation, should be pointed out. Unpaired buried charges are energetically too expensive to exit in the hydrophobic core. Isolated hydrogen bond donors and acceptors also exert negative effects, but they are not as expensive as the unpaired buried charges, with costs of a few kcal/mol. Therefore, statistical analyses of protein 3D structures reveal only rare instances of isolated hydrogen bond donors and acceptors. This must be the main reason why alpha-helices and beta-sheets are only observed in protein cores as the backbone structures. Such secondary structures do not leave any backbone hydrogen donors or acceptors unpaired, because of their intrinsically regular packing. Otherwise, it might be very difficult to construct a backbone structure, in which all the backbone amide and carbonyl groups had their own hydrogen bond partners in the protein core. There are two theoretical approaches to protein electrostatics, the macroscopic or continuum model, and the microscopic or molecular model. As described in this article, the macroscopic model has inherent problems because the protein-solvent system is very hetergeneous from the physical point of view...

Protein Structure-Based Combinatorial Chemistry: Discovery of Non-Peptide Binding Elements to Src SH3 Domain

Abstract: Small molecule ligands can be used to cause a conditional loss or gain of function of their protein receptors and, therefore, can be viewed as equivalents of conditional alleles.1-3 In order to extend their use, methods to identify such ligands de novo are required. We have previously reported the use of biased combinatorial libraries to discover peptide ligands to proteins,4 and the coupled use of combinatorial chemistry and structural biology to understand the nature of protein-ligand interactions.5-7 More recently, we have been exploring whether the knowledge of protein structure can facilitate the design of monomers and linking elements leading to vast numbers of potential ligands targeted to a particular protein. We now report a first illustration of this strategy resulting in the discovery of novel and specific ligands containing non-peptide structural elements. Structural investigations of SH3-peptide complexes have revealed that SH3 domains bind peptide ligands in either of two orientations (classes I and II; these differ in the directionality of the backbone amides5,6) involving the three pockets depicted in Figure 1.4-7 We designed a library of ligands predisposed to adopt the class I orientation by attaching a common lowaffinity (Kd > 1 mM) biasing sequence PLPPLP (P ) Pro, L ) Leu) to a solid support. This sequence was expected to fill the two pockets (labeled 1 and 2) that bind Leu-Pro dipeptides. Furthermore, structural analyses show that the N-terminal proline should be positioned to orient elements attached to its pyrrolidine nitrogen into the third pocket (labeled 3), which is lined by the nSrc and RT loops common to all SH3 domains and is the primary determinant of ligand specificity.8 We synthesized an encoded9 combinatorial library derived from 32 monomers incorporated during three consecutive cycles of split-and-pool synthesis10,11 following the synthesis of the common PLPPLP sequence (synthesized in the C to N direction) and terminating with one of 32 capping reagents (Figure 2). We purposefully incorporated an encoded blank (“skip-codon”)12 during monomer and cap incorporation in order to increase library diversity significantly by creating sublibraries with deletions at any one or more of the three monomer and one cap sites.

CD Spectroscopy and the Helix-Coil Transition in Peptides and Polypeptides

Abstract: The proposal by Pauling and his coworkers (1951) of an atomic model for the structure of the alpha helix stimulated research in several areas of protein chemistry It excited chemists as few discoveries have before or since, giving impetus to structural modeling efforts that resulted in the structure of DNA 2 years later, and in a whole new field of structural biology within two decades Pauling’s feat pointed out the importance of understanding the conformation of the peptide group itself, rather than building models based on idealized helical structures Working on the same problem, Bragg et al (1950) failed to produce a structure of comparable elegance because they were unaware the peptide bond was planar (Crick, 1988) The alpha helix could be identified in the diffraction patterns from crystals of the globular proteins myoglobin and hemoglobin, as well as in the classical “α” patterns from fibrous proteins like keratin and synthetic polypeptides, poly(γ-l-glutamate) being the first to show the α pattern (Elliott, 1967)

Nucleic Acid Structures, Energetics, and Dynamics

Abstract: In 1953 DNA was shown to be a double helix of hydrogen-bonded complementary bases. Since then, knowledge of nucleic acid structures, thermodynamic stabilities, and dynamics of conformational changes has grown exponentially. This knowledge has led to the development of the biotechnology industry, the identification of plants and animals from a few cells, and many advances in the diagnosis and treatment of diseases. All the methods of physical chemistry have been used to characterize the primary structures (sequences), the secondary structures (base pairing), and the tertiary structures (folded 3D conformations) of the nucleic acids. The interactions of nucleic acids with themselves, with proteins, and with small-molecule ligands control their many functions. Novel methods are being developed to probe the structures and functions of nucleic acids. These include methods to study a single molecule and methods to select, amplify, and characterize one sequence among 1017 different sequences.

Peripheral Membrane Proteins

Abstract: The founding principle behind structural biology is that form equals function. Nowhere is this relation more apparent than in proteins that operate in environments in which aqueous and lipophilic phases meet. This biphasic environment strongly influences both the structure and function of these proteins. Membrane proteins can be classified as integral or peripheral, depending upon the nature of the protein-membrane association. For proteins in the former category, the membrane is an integral part of their structures, which are greatly perturbed by disruption of the membrane by detergents. The extent of interaction with the lipid bilayer is usually obvious by simple inspection of the protein structure because of what is known about lipid physical chemistry, i.e., that there is a large energy cost to burying uncompensated polar or charged protein residues in a strongly hydrophobic milieu. For example, the membrane-embedded portions of transmembrane proteins, such as bacterial reaction centers and porins, are localized by the extensive hydrophobic regions, which are frequently bordered by aromatic side-chains, found on the surfaces of these proteins. For these transmembrane proteins, whose functional roles are to transfer energy or substances from one aqueous pool to another across a nonaqueous barrier, the membrane serves to organize the protein structure. In contrast to integral membrane proteins, those classified as peripheral can be released from their membrane attachment through gentler means, without disruption of either membrane or protein structure.

Applications of Multidimensional Solid-State NMR Spectroscopy to Membrane Proteins

Abstract: A singular challenge in structural biology is the experimental determination of the structures of membrane proteins. Because these proteins are difficult to crystallize there are few examples with structures determined by X-ray diffraction (Deisenhofer et al., 1985; Weiss et al. 1991; Iwata et al., 1995). Multidimensional solution NMR methods are difficult to apply to membrane proteins because of the slow reorientation rates and broad resonance linewidths that accompany solubilisation in detergent micelles. Proteins in the other well characterized model membrane environment of lipid bilayers are even less well suited for solution NMR methods because the individual protein molecules are effectively immobilized when complexed with phospholipids. However, it is useful to keep in mind that NMR studies of membrane proteins are formidable because of the motional properties of the samples rather than any intrinsic properties of the proteins themselves and that solid state NMR spectroscopy is fully capable of overcoming the difficulties resulting from the very slow reorientation rates (Opella, 1994).

Computational Refinement Through Solid State NMR and Energy Constraints of a Membrane Bound Polypeptide

Abstract: The determination of macromolecular structures in anisotropic environments such as membranes is vital to the field of structural biology. While solid state nuclear magnetic resonance spectroscopy (SSNMR) methods have been demonstrated for obtaining three dimensional structures of membrane bound polypeptides (Cross and Opella, 1983; Ketchem et al, 1993; Opella et al, 1987), computational refinement methods are needed for optimally utilizing these constraints in such a molecular environment. Methods for structural determination and refinement of macromolecules in solution have fully evolved (Briinger et al, 1986; Clore et al, 1985; Havel and Wuthrich, 1985), but the nature of the constraints obtained for membrane proteins are such that a new refinement procedure must be developed. Described here is such a technique that has the ability to optimize the structure of a membrane protein in order to best represent the experimental data and determine its high-resolution structure.

Developments in NMR Structure Determination of Nucleic Acids

Abstract: The spectacular developments in high resolution NMR, during the last five to ten years, have established the technique as one of the most important methods in the field of structural biology. With the aid of NMR spectroscopy structures of biomolecules in aqueous solutions can be determined; the other powerful technique playing a key role in structural biology, X-ray diffraction, needs molecules in the crystal form in order to elucidate their structures. In the present contribution some of the NMR methods used to determine nucleic acid structures will be discussed. Compared to protein structure determination, by means of NMR, the elucidation of nucleic acid structures has its peculiar difficulties and as a result has lagged somewhat behind. One reason is that functional nucleic acid molecules are normally very large and not accessible to structure determination by the high resolution techniques X-ray crystallography and NMR spectroscopy. It has been found, however, that in these molecules particular regions may adopt highly intricate folding patterns which are functionally very important. Examples are the ribozyme [Pley et al. (1994); Scott et al., (1995)] and pseudoknot motifs in RNA [Pleij, (1990)] and the H-DNA triple helices [Frank-Kamenetskii and Mirkin, (1995)] and hairpins in DNA [Hilbers et al. (1994)]. DNA and RNA fragments containing these folding motifs are amenable to high resolution structural studies and these investigations form the basis for a reliable description of the structure function relationships in these molecules. Other reasons for the slower progress in NMR structure determination of nucleic acids follow from a further comparison between nucleic acid and protein structural characteristics.

NMR Structure Determination and Rational Drug Design

Abstract: In biological and biomedical research, the area of structural biology, in particular atomic resolution studies of the three-dimensional structure of biological macromolecules and their intermolecular interactions, has never before had as central a role and enjoyed as much popularity as today. This is mainly due to the facts that with the use of DNA recombination and chemical synthesis, a virtually unlimited array of different polypeptide sequences can be generated, and that the relations of the resulting primary structures with corresponding biological functions can only be rationalized through knowledge of the three-dimensional structure. In addition to its key role in academic institutions, structure determination of proteins, nucleic acids and other classes of biomolecules has therefore become a discipline that is actively pursued also in profit-oriented organizations, especially in the major pharmaceutical companies. Although X-ray diffraction with single crystals has for two decades, until 1984, been unique in its potential for efficient determination of three-dimensional molecular structures at atomic resolution, it presently shares this role with nuclear magnetic resonance (NMR) spectroscopy in solution. During the period 1990–1993, 607 new X-ray crystal structures, 179 new NMR solution structures, and 2 structures determined by other methods were published (Hendrickson and Wuthrich, 1991; 1992; 1993; 1994). This article presents a description of the NMR approach for three-dimensional structure determination of biological macromolecules and a discussion of the impact of molecular structural biology in biomedical research.

Protein-DNA Interaction from NMR and Monte Carlo Docking

Abstract: An essential step in the regulation of gene expression is the binding of a regulatory protein to a specific DNA sequence in the promotor region of the gene. The understanding of protein-DNA recognition is, therefore, a major theme in structural biology. Much progress has been made since the early ’80s when the first structures of bacterial DNA-binding proteins and protein-DNA complexes were solved by X-ray crystallography (for reviews see Steitz (1990), Pabo and Sauer (1992) and Travers (1993)). NMR started to contribute around 1985 with the structure elucidation of the lac repressor headpiece (Kaptein et al., 1985) and a low resolution structure of the headpiece-operator complex (Boelens et al., 1987). These first prokaryotic DNA-binding proteins all contained the helix-turn-helix motif as the DNA-binding subdomain. However, subsequently a large number of other structural motifs has been characterized including zinc-fingers, leucine zippers, helix-loop-helix proteins and β-sheet DNA binding proteins.