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M Carrion-Vazquez

Publications and source records attributed to M Carrion-Vazquez.

13 recordsLinked to original sources
PubMed2001

Multiple conformations of PEVK proteins detected by single-molecule techniques.

An important component of muscle elasticity is the PEVK region of titin, so named because of the preponderance of these amino acids. However, the PEVK region, similar to other elastomeric proteins, is thought to form a random coil and therefore its structure cannot be determined by standard techniques. Here we combine single-molecule electron microscopy and atomic force microscopy to examine the conformations of the human cardiac titin PEVK region. In contrast to a simple random coil, we have found that cardiac PEVK shows a wide range of elastic conformations with end-to-end distances ranging from 9 to 24 nm and persistence lengths from 0.4 to 2.5 nm. Individual PEVK molecules retained their distinctive elastic conformations through many stretch-relaxation cycles, consistent with the view that these PEVK conformers cannot be interconverted by force. The multiple elastic conformations of cardiac PEVK may result from varying degrees of proline isomerization. The single-molecule techniques demonstrated here may help elucidate the conformation of other proteins that lack a well-defined structure.

Connectin↗
PubMed2001

Stepwise unfolding of titin under force-clamp atomic force microscopy.

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.

Animals↗
PubMed2000

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals.

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.

Animals↗
PubMed2000

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Elasticity↗
PubMed2000

Point mutations alter the mechanical stability of immunoglobulin modules.

Immunoglobulin-like modules are common components of proteins that play mechanical roles in cells such as muscle elasticity and cell adhesion. Mutations in these proteins may affect their mechanical stability and thus may compromise their function. Using single molecule atomic force microscopy (AFM) and protein engineering, we demonstrate that point mutations in two beta-strands of an immunoglobulin module in human cardiac titin alter the mechanical stability of the protein, resulting in mechanical phenotypes. Our results demonstrate a previously unrecognized class of phenotypes that may be common in cell adhesion and muscle proteins.

Amino Acid Substitution↗
PubMed1999

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.

Biomechanical Phenomena↗
PubMed1999

The micro-mechanics of single molecules studied with atomic force microscopy.

The atomic force microscope (AFM) in its force-measuring mode is capable of effecting displacements on an angstrom scale (10 A = 1 nm) and measuring forces of a few piconewtons. Recent experiments have applied AFM techniques to study the mechanical properties of single biological polymers. These properties contribute to the function of many proteins exposed to mechanical strain, including components of the extracellular matrix (ECM). The force-bearing proteins of the ECM typically contain multiple tandem repeats of independently folded domains, a common feature of proteins with structural and mechanical roles. Polysaccharide moieties of adhesion glycoproteins such as the selectins are also subject to strain. Force-induced extension of both types of molecules with the AFM results in conformational changes that could contribute to their mechanical function. The force-extension curve for amylose exhibits a transition in elasticity caused by the conversion of its glucopyranose rings from the chair to the boat conformation. Extension of multi-domain proteins causes sequential unraveling of domains, resulting in a force-extension curve displaying a saw tooth pattern of peaks. The engineering of multimeric proteins consisting of repeats of identical domains has allowed detailed analysis of the mechanical properties of single protein domains. Repetitive extension and relaxation has enabled direct measurement of rates of domain unfolding and refolding. The combination of site-directed mutagenesis with AFM can be used to elucidate the amino acid sequences that determine mechanical stability. The AFM thus offers a novel way to explore the mechanical functions of proteins and will be a useful tool for studying the micro-mechanics of exocytosis.

Animals↗
PubMed1999

Atomic force microscopy captures length phenotypes in single proteins.

We use single-protein atomic force microscopy techniques to detect length phenotypes in an Ig module. To gain amino acid resolution, we amplify the mechanical features of a single module by engineering polyproteins composed of up to 12 identical repeats. We show that on mechanical unfolding, mutant polyproteins containing five extra glycine residues added to the folded core of the module extend 20 A per module farther than the wild-type polyproteins. By contrast, similar insertions near the N or C termini have no effect. Hence, our atomic force microscopy measurements readily discriminate the location of the insert and measure its size with a resolution similar to that of NMR and x-ray crystallography.

Connectin↗
PubMed1999

Mechanical and chemical unfolding of a single protein: a comparison.

Is the mechanical unraveling of protein domains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero force. We compare this with chemical unfolding rates for untethered modules extrapolated to 0 M denaturant. The unfolding rates obtained by the two methods are the same. Furthermore, the transition state for unfolding appears at the same position on the folding pathway when assessed by either method. These results indicate that mechanical unfolding of a single protein by AFM does indeed reflect the same event that is observed in traditional unfolding experiments. The way is now open for the extensive use of AFM to measure folding reactions at the single-molecule level. Single-molecule AFM recordings have the added advantage that they define the reaction coordinate and expose rare unfolding events that cannot be observed in the absence of chemical denaturants.

Calmodulin-Binding Proteins↗
PubMed1999

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.

Entropy↗
PubMed1999

Single protein misfolding events captured by atomic force microscopy.

Using single protein atomic force microscopy (AFM) techniques we demonstrate that after repeated mechanical extension/relaxation cycles, tandem modular proteins can misfold into a structure formed by two neighboring modules. The misfolding is fully reversible and alters the mechanical topology of the modules while it is about as stable as the original fold. Our results show that modular proteins can assume a novel misfolded state and demonstrate that AFM is able to capture, in real time, rare misfolding events at the level of a single protein.

Connectin↗