Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

The combinatorial distance geometry method for the calculation of molecular conformation. II. Sample problems and computational statistics

The performance of a branch and bound algorithm for molecular energy minimization is evaluated on a variety of test problems. Although not at present efficient enough for use in most practical situations, we show that it has distinct advantages over more conventional methods of global minimization. In addition, this study illustrates the technique on which the present algorithm is based, and the problems which must be overcome in developing an efficient algorithm based on similar principles.     

Correlation of sequence and tertiary structure in globular proteins

The x-ray crystal structures of 19 selected proteins are examined empirically for correlations between the amino acid sequence and long-range, tertiary conformation. There is clear evidence for preferential associations of certain types of amino acids, particularly among the hydrophobic aliphatic, aromatic, and cysteine residues. However, the likelihoods of forming these residue-pair contacts are all less than 12%, so packing and geometric requirements must often take precedent over energetic considerations. The prediction of long-range contacts is not substantially improved by taking into account the sequentially previous residues. The analysis of atom–atom contacts shows a similar lack of predictive ability, but the results show that a good approximation to the interresidue energy function must include different types of interactions at two or three different sites on some amino acids. Backbone–backbone long-range interactions are relatively rare and nonspecific, whereas some “polar” side chains form hydrogen bonds from the polar groups while occasionally forming hydrophobic contacts with the remainder of the chain.     

Prediction of protein folding from amino acid sequence over discrete conformation spaces

Predicting the three-dimensional structure of a protein given only its amino acid sequence is a long-standing goal in computational chemistry. In the thermodynamic approach, one needs a potential function of conformation that resembles the free energy of the real protein to the extent that the global minimum of the potential is attained by the native conformation and no other. In practice, this has never been achieved with certainty because even with greatly simplified representations of the polypeptide chain, there are an astronomical number of local minima to examine. If one chooses instead a protein representation with only a large but manageable number of discrete conformations, then the global preference of the potential for the native can be directly verified. Representing a protein as a walk on a two-dimensional square lattice makes it easy to see that simple functions of the interresidue contacts are sufficient to globally favor a given "native" conformation, as long as it is a compact, globular structure. Explicit representation of the solvent is not required. Another more realistic way to confine the conformational search to a finite set is to draw alternative conformations from fragments of larger proteins having known crystal structure. Then it is possible to construct a simple function of interresidue contacts in three dimensions such that only 8 proteins are required to determine the adjustable parameters, and the native conformations of 37 other proteins are correctly preferred over all alternative conformations. The deduced function favors short-range backbone-backbone contacts regardless of residue type and long-range hydrophobic associations. Interactions over long distances, such as electrostatics, are not required.     

Protein densities.

The calculation of protein densities from atomic coordinates is not straight-forward and requires very careful attention to the determination of the protein-solvent boundary. Interior densities are more readily obtained and are in reasonable agreement with those estimated from solvent accessibility studies. The interior of globular proteins has very significant density inhomogenities on a scale of 100--1000 A3. The interior densities range from less than 0.5 g/cm3 to over 3 g/cm3. The low local densities are primarily associated with clusters of nonpolar sidechains while the high local density regions arise from the protein backbone secondary structures: helices and beta sheets. We show a rough correlation between local density and local polarity.     

Potential energy function for continuous state models of globular proteins.

One of the approaches to protein structure prediction is to obtain energy functions which can recognize the native conformation of a given sequence among a zoo of conformations. The discriminations can be done by assigning the lowest energy to the native conformation, with the guarantee that the native is in the zoo. Well-adjusted functions, then, can be used in the search for other (near-) natives. Here the aim is the discrimination at relatively high resolution (RMSD difference between the native and the closest nonnative is around 1 A) by pairwise energy potentials. The potential is trained using the experimentally determined native conformation of only one protein, instead of the usual large survey over many proteins. The novel feature is that the native structure is compared to a vastly wider and more challenging array of nonnative structures found not only by the usual threading procedure, but by wide-ranging local minimization of the potential. Because of this extremely demanding search, the native is very close to the apparent global minimum of the potential function. The global minimum property holds up for one other protein having 60% sequence identity, but its performance on completely dissimilar proteins is of course much weaker.