Mechanical unfolding intermediates in titin modules

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.     

Abundant gene conversion between arms of palindromes in human and ape Y chromosomes

Eight palindromes comprise one-quarter of the euchromatic DNA of the male-specific region of the human Y chromosome, the MSY. They contain many testis-specific genes and typically exhibit 99.97% intra-palindromic (arm-to-arm) sequence identity. This high degree of identity could be interpreted as evidence that the palindromes arose through duplication events that occurred about 100,000 years ago. Using comparative sequencing in great apes, we demonstrate here that at least six of these MSY palindromes predate the divergence of the human and chimpanzee lineages, which occurred about 5 million years ago. The arms of these palindromes must have subsequently engaged in gene conversion, driving the paired arms to evolve in concert. Indeed, analysis of MSY palindrome sequence variation in existing human populations provides evidence of recurrent arm-to-arm gene conversion in our species. We conclude that during recent evolution, an average of approximately 600 nucleotides per newborn male have undergone Y-Y gene conversion, which has had an important role in the evolution of multi-copy testis gene families in the MSY.     

High mutation rates have driven extensive structural polymorphism among human Y chromosomes

Although much structural polymorphism in the human genome has been catalogued, the kinetics of underlying change remain largely unexplored. Because human Y chromosomes are clonally inherited, it has been possible to capture their detailed relationships in a robust, worldwide genealogical tree. Examination of structural variation across this tree opens avenues for investigating rates of underlying mutations. We selected one Y chromosome from each of 47 branches of this tree and searched for large-scale variation. Four chromosomal regions showed extensive variation resulting from numerous large-scale mutations. Within the tree encompassed by the studied chromosomes, the distal-Yq heterochromatin changed length > or = 12 times, the TSPY gene array changed length > or = 23 times, the 3.6-Mb IR3/IR3 region changed orientation > or = 12 times and the AZFc region was rearranged > or = 20 times. After determining the total time spanned by all branches of this tree (approximately 1.3 million years or 52,000 generations), we converted these mutation counts to lower bounds on rates: > or = 2.3 x 10(-4), > or = 4.4 x 10(-4), > or = 2.3 x 10(-4) and > or = 3.8 x 10(-4) large-scale mutations per father-to-son Y transmission, respectively. Thus, high mutation rates have driven extensive structural polymorphism among human Y chromosomes. At the same time, we found limited variation in the copy number of Y-linked genes, which raises the possibility of selective constraints.     

Nanospring behaviour of ankyrin repeats

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.     

A specialized mitochondrial molecular chaperone system:¶A role in formation of Fe/S centers

Mitochondria contain a specialized system of molecular chaperones that plays a critical role in the biogenesis of Fe/S centers. This Hsp70:J-protein system shows many similarities to the system found in bacteria, but the precise role of neither chaperone system has been defined. However, evidence to date suggests an interaction with the scaffold protein on which a transient Fe/S center is assembled, and thus implies a role in either assembly of the center or its transfer to recipient proteins.     

Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.