Showing papers by "Chris Sander published in 1989"

Polarity as a criterion in protein design

Abstract: Hypothetical proteins can be tested computationally by determining whether or not the designed sequence-structure pair has the characteristics of a typical globular protein. We have developed such a test by deriving quantities with approximately constant value for all globular proteins, based on empirical analysis of the exposed and buried surfaces of 128 structurally known proteins. The characteristic quantities that best appear to segregate badly designed or deliberately misfolded proteins from their properly folded natural relatives are the polar fraction of side chains on the protein surface and, independently, in the protein interior. Three of the seven hypothetical structures tested here can be rejected as having too many polar side-chain groups in the interior or too few on the protein surface. In addition, a recently designed nutritional protein is identified as being very much unlike globular proteins. These database-derived characteristic quantities are useful in screening designed proteins prior to experiment and may be useful in screening experimentally determined (X-ray, NMR) protein structures for possible errors.

How to determine protein secondary structure in solution by Raman spectroscopy: practical guide and test case DNase I

Abstract: For many proteins available in large (milligram) quantities, a three-dimensional structure determination by X-ray or NMR methods is very difficult, impossible, or too costly. In these cases, spectroscopic determination of secondary structure content can be a valuable source of partial information about protein structure in solution. In particular, Raman spectroscopy can be used to determine to fair accuracy the helix and sheet content of a globular protein. However, technical difficulties have hampered the routine application of the method: (1) The large background signal of aqueous solvent in the amide I region is difficult to subtract accurately. (2) The reference data set of Raman spectra of proteins with known crystal structure is incomplete, and the assignment of secondary structure in a known crystal structure is not unambiguous. (3) The mathematical problem of extracting structure information from the spectra is ill determined; i.e., there are many apparently satisfactory solutions for a given spectrum. We have now partly solved and partly sidestepped these problems by improving and simplifying existing methods. Here, we give a step-by-step outline of a procedure intended for routine determination of the percentage of a-helix and @-sheet from the amide I Raman spectra of proteins in solution. Its main features are (a) an uncom- plicated procedure for solvent subtraction, aided by use of a divided spinning cell technique, (b) a numerically stable data handling algorithm, and (c) a clear statement of expected accuracy. In our hands, using the reference spectra of Williams (1 983), helix content can be determined to an accuracy of 6 percentage points (largest error 12%) and @-sheet content to an accuracy of 5 percentage points (largest error 7%). However, the experimental distinction between parallel and antiparallel 0-sheet does not appear possible without a significant expansion of the set of reference proteins. As a test we have measured the Raman spectrum of DNase I, a known structure treated as unknown, and derive 14% a-helix and 22% 0-sheet content, compared to X-ray derived values of 20% helix and 25% sheet (hydrogen bonds per 100 residues). The error, -6% for helix and -3% for sheet content, is typical. The method can be a tool for checking the structural purity of genetically engineered proteins, detecting major structural alterations of mutant proteins, and providing a priori information as input to predictions of protein structure.

Thermitase, a thermostable subtilisin: Comparison of predicted and experimental structures and the molecular cause of thermostability

Abstract: The subtilisin family of proteases has four members of known sequence and structure: subtilisin Carlsberg, subtilisin novo, proteinase K, and thermitase. Using thermitase as a test case, we ask two questions. How good are methods for model building a three-dimensional structure of a protein based on sequence homology to a known structure? And what are the molecular causes of thermostability? First, we compare predicted models of thermitase, refined by energy minimization and varied by molecular dynamics, with the preliminary crystal structure. The predictions work best in the conserved structural core and less well in seven loop regions involving insertions and deletions relative to subtilisin. Here, variation of loop regions by molecular dynamics simulation in vacuo followed by energy minimization does not improve the prediction since we find no correlation between in vacuo energy and correctness of structure when comparing local energy minima. Second, in order to identify the molecular cause of thermostability we confront hypotheses derived by calculation of the details of interatomic interactions and estimates of hydrophobic interactions with inactivation experiments. As a result, we can exclude salt bridges and hydrophobic interactions as main causes of thermostability. Based on a combination of theoretical and experimental evidence, the unusually tight binding of calcium by thermitase emerges as the most likely single influence responsible for its increased thermostability.

Conservation of residue interactions in a family of Ca-binding proteins

Abstract: In the TNC family of Ca-binding proteins (calmodulin, parvalbumin, intestinal calcium binding protein and troponin C) approximately 70 well-conserved amino acid sequences and six crystal structures are known. We find a clear correlation between residue contacts in the structures and residue conservation in the sequences: residues with strong sidechain-sidechain contacts in the three-dimenesional structure tend to be the more conserved in the sequence. This is one way to quantify the intuitive notion of the importance of sidechain interactions for maintaining protein three-dimensional structure in evolution and may usefully be taken into account in planning point mutations in protein engineering.

Identification of Guanine-Nucleotide Binding Proteins in Plants: Structural Analysis and Evolutionary Comparison of the Ras-Related Ypt-Gene Family from Zea Mays

Abstract: The development of plants is regulated by five types of hormones known as auxins, cytokinins, gibberellins, abscisic acid and ethylene (Phillips, 1971). Only little is known about the mechanisms of action of these hormones at the cellular and molecular level. Genetic analysis of mutants with increased resistance to growth inhibiting concentrations of hormones argues for the presence of receptor like functions in plants (King, 1988). Biochemical evidence suggests that the action of auxins may be mediated after binding to plasmalemma located receptors (for review see: Davies, 1988). However, our current picture on other elements in the plant cell, involved in the transmission of this signal to its final biological target, is only slowly emerging. Recent evidence strengthens the importance of the calcium signal in plant cells (Marme, 1983; Hepler & Wayne, 1985). Transient changes in cytoplasmic calcium levels coupled to external stimuli such as light and phytohormones can modulate numerous developmental responses (Hepler & Wayne, 1985).