Advances in Protein Chemistry最新文献

The amino acid sequences of increasingly large proteins have been determined in recent years, and it has become more and more apparent that within these sequences nature has employed only a finite number of structural?functional motifs. These may be strung along the sequence in tandem and, in some cases, several hundred times. In other instances, the positions of the motifs show little obvious order as regards to their relative linear arrangement within the sequence. The observed sequence repeats have been shown to vary in size over at least two orders of magnitude. It is shown here that the repeats can readily be classified on the basis of character, and five distinct groups have been identified. The first of these (Type A) represents those motifs that are fixed in length and conserved absolutely in sequence (>99%); the second (Type B) includes motifs that are also fixed in length, but where absolute sequence conservation occurs only in some positions of the repeat. The third category (Type C) contains fixed length motifs, but the character of only some of the positions in the motif is maintained. The fourth group (Type D) includes motifs that have nonintegral lengths. The fifth class (Type E) contains motifs, often displaying some variations in their lengths even within a single species, which maintain a discrete structural form related directly to their function. Examples are presented for each category of repeat, and these are drawn almost exclusively from the fibrous proteins and those proteins that are normally associated with them in vivo.

alpha-Helical coiled coils are versatile protein domains, supporting a wide range of biological functions. Their fold is probably better understood than that of any other protein; indeed, uniquely among folds, their structure can be computed from a set of parametric equations. Here, we review the principles of coiled-coil structure, the determinants of their folding and stability, and the diversity of structural forms they assume.

The molecular conformation of the collagen triple helix confers strict amino acid sequence constraints, requiring a (Gly-X-Y)(n) repeating pattern and a high content of imino acids. The increasing family of collagens and proteins with collagenous domains shows the collagen triple helix to be a basic motif adaptable to a range of proteins and functions. Its rodlike domain has the potential for various modes of self-association and the capacity to bind receptors, other proteins, GAGs, and nucleic acids. High-resolution crystal structures obtained for collagen model peptides confirm the supercoiled triple helix conformation, and provide new information on hydrogen bonding patterns, hydration, sidechain interactions, and ligand binding. For several peptides, the helix twist was found to be sequence dependent, and such variation in helix twist may serve as recognition features or to orient the triple helix for binding. Mutations in the collagen triple-helix domain lead to a variety of human disorders. The most common mutations are single-base substitutions that lead to the replacement of one Gly residue, breaking the Gly-X-Y repeating pattern. A single Gly substitution destabilizes the triple helix through a local disruption in hydrogen bonding and produces a discontinuity in the register of the helix. Molecular information about the collagen triple helix and the effect of mutations will lead to a better understanding of function and pathology.

Coiled-coil proteins, collagen, and elastomers together comprise an important subset of the fibrous proteins. The former group-the alpha-fibrous coiled-coil proteins-are widely distributed in nature and, indeed, the characteristic heptad motif has been recognized as an oligomerisation motif in fibril-forming collagens. This volume has selected a number of the alpha-fibrous proteins for detailed discussion, including intermediate filament proteins, the spectrin superfamily, and fibrin?fibrinogen. Of particular interest is the growing realization that the design principles governing the structures of these coiled-coil proteins are now largely discernible and can be specified with a high degree of confidence, due in large part to the wealth of crystal structure data now available. Within the connective tissues covered in this volume, two constituents of defining importance mechanically are the collagen fibrils?networks and the elastic fibers. Crystal structures of collagen peptides have been published and are described. The effects of the precise sequence of the distinct constituent triplets on molecular conformation have also become clearer. The ultrastructures of connective tissues are largely defined by the spatial arrangement of the collagen fibrils and networks, and this is elucidated here in some detail. The elastic fibers with their elastin cores and fibrillin-containing microfibril palisades are also described. A theme underlying all of the proteins discussed in this volume is the significantly increased effort to characterize the structures and functions of mutants. Some of these occur naturally and lead to various disease states, while others have been genetically engineered in order to study design principles.

Microtubules are the intracellular tracks for two classes of motor proteins: kinesins and dyneins. During the past few years, the motor domain structures of several kinesins from different organisms have been determined by X-ray crystallography. Compared with kinesins, dyneins are much larger proteins and attempts to crystallize them have failed so far. Structural information about these proteins comes mostly from electron microscopy. In this chapter, we mainly focus on the crystal structures of kinesin motor domains.

Nematode sperm provide a simple and specialized system for studying the molecular mechanism of amoeboid cell motility. Locomotion is generated by the assembly dynamics of their cytoskeleton, which is based on the major sperm protein (MSP). Protrusive force is generated at the leading edge of the lamellipod by MSP filament formation and bundling, whereas the contractile force that drags the rearward cell body forward is generated by cytoskeleton disassembly. The dynamics of the system can be reconstituted in vitro using cell-free extracts of Ascaris sperm, in which vesicles derived from the leading edge of the cell can be either pushed or pulled. The addition of ATP to the cell-free extract initiates MSP filament polymerization and bundling immediately behind the vesicle, and the expansion of the resulting gel pushes the vesicle at rates comparable to those seen in living cells. In contrast, the addition of Yersinia tyrosine phosphatase generates depolymerization and gel contraction that pulls the vesicles. Overall, nematode sperm motility illustrates that cell locomotion can be generated by cytoskeletal dynamics alone without the use of myosin-like motor proteins.

The contribution of backbone-backbone hydrogen bonds (H-bonds) to protein folding energetics has been controversial. This is due, at least in part, to the inability to perturb backbone-backbone H-bonds by traditional methods of protein mutagenesis. Recently, however, protein backbone mutagenesis has become possible with the development of chemical and biological methods to replace individual amides in the protein backbone with esters. Here, we review the use of amide-to-ester mutation as a tool to evaluate the contribution of backbone-backbone H-bonds to protein folding kinetics and thermodynamics.

Proteins and peptides in solution or in vivo share properties with both liquids and solids. More often than not, they are studied using the liquid paradigm rather than that of a solid. Studies of molecular crystals illustrate how the use of a solid paradigm may change the way that we consider these important molecules. Cooperative interactions, particularly those involving H-bonding, play much more important roles in the solid than in the liquid paradigms, as molecular crystals clearly illustrate. Using the solid rather than the liquid paradigm for proteins and peptides includes these cooperative interactions while application of the liquid paradigm tends to ignore or minimize them. Use of the solid paradigm has important implications for basic principles that are often implied about peptide and protein chemistry, such as the importance of entropy in protein folding and the nature of the hydrophobic effect. Understanding the folded states of peptides and proteins (especially alpha-helices) often requires the solid paradigm, whereas understanding unfolded states does not. Both theoretical and experimental studies of the energetics of protein and peptide folding require comparison to a suitable standard. Our perspective on these energetics depends on the reasonable choice of reference. The use of multiple reference states, particularly that of component amino acids in the gas phase, is proposed.

Fibrillin microfibrils are widely distributed extracellular matrix assemblies that endow elastic and nonelastic connective tissues with long-range elasticity. They direct tropoelastin deposition during elastic fibrillogenesis and form an outer mantle for mature elastic fibers. Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the ciliary zonules of the eye. Mutations in fibrillin-1-the principal structural component of microfibrils-cause Marfan syndrome, a heritable disease with severe aortic, ocular, and skeletal defects. Isolated fibrillin-rich microfibrils have a complex 56 nm "beads-on-a-string" appearance; the molecular basis of their assembly and elastic properties, and their role in higher-order elastic fiber formation, remain incompletely understood.