The misfolding of serpins is linked to several genetic disorders including emphysema, thrombosis, and dementia. During folding, inhibitory serpins are kinetically trapped in a metastable state in which a stretch of residues near the C terminus of the molecule are exposed to solvent as a flexible loop (the reactive center loop). When they inhibit target proteases, serpins transition to a stable state in which the reactive center loop forms part of a six-stranded β-sheet. Here, we use hydrogen-deuterium exchange mass spectrometry to monitor region-specific folding of the canonical serpin human α 1 -antitrypsin (α 1 -AT). We find large differences in the folding kinetics of different regions. A key region in the metastable → stable transition, β-strand 5A, shows a lag phase of nearly 350 s. In contrast, the "B-C barrel" region shows no lag phase and the incorporation of the C-terminal residues into β-sheets B and C is largely complete before the center of β-sheet A begins to fold. We propose this as the mechanism for trapping α 1 -AT in a metastable form. Additionally, this separation of timescales in the folding of different regions suggests a mechanism by which α 1 -AT avoids polymerization during folding.hydrogen exchange | misfolding disease | protein folding T he misfolding and polymerization of serpins is linked to a number of inherited diseases including liver cirrhosis, emphysema, and dementia (1). In the most common serpin-linked disease, α 1 -antitrypsin deficiency, α 1 -antitrypsin (α 1 -AT) is trapped in a misfolded polymerization prone state during processing in the endoplasmic reticulum. Accumulation of polymers ultimately leads to apoptosis. Unlike in amyloid diseases, however, it is not thought that serpins undergo a compete change in their secondary and tertiary structures upon polymerization. Instead, serpin misfolding is thought to be related to their unusual inhibitory mechanism.The lowest free energy structure of α 1 -AT is shown in Fig. 1B. However, the structure shown in Fig. 1A is the structure that α 1 -AT spontaneously adopts during folding. Alpha-1 antitrypsin, like other inhibitory serpins, is trapped in a metastable state (2). The α 1 -AT fold consists of three β-sheets (A, B, and C) and nine α-helices (A-I). In the metastable form, residues 345-360 are exposed to solvent and comprise the reactive center loop (RCL) that is cleaved by target proteases, whereas in the stable form, these same residues form the fourth strand of β-sheet A. Cleavage by target proteases triggers a conformational change in which the N-terminal portion of the RCL spontaneously inserts into β-sheet A, carrying the bound protease (covalently linked as an acylenzyme intermediate) along with it.Evidence suggests that serpin polymerization is an intermolecular version of the above-described intramolecular process. The two most prominent models for serpin polymerization propose that polymers are formed either through the insertion of the RCL of one serpin into the A β-sheet of another, or through a domain swap mechani...

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.

Knowledge of the structure and dynamics of proteins and protein assemblies is critical both for understanding the molecular basis of physiological and patho-physiological processes and for guiding drug design. While X-ray crystallography and nuclear magnetic resonance spectroscopy are both excellent techniques for this purpose, both suffer from limitations, including the requirement for high quality crystals and large amounts of material. Recently, hydrogen/deuterium exchange measured using mass spectrometry (HXMS) has emerged as a powerful new tool for the study of protein structure, dynamics and interactions in solution. HXMS exploits the fact that backbone amide hydrogens can exchange with deuterium when a protein is incubated in D(2)O, and that the rate of the exchange process is highly dependent on the local structural environment. Several features of HXMS make it an especially attractive approach, including small sample requirements and the ability to study extremely large protein assemblies that are not amenable to other techniques. Here, we provide an overview of HXMS and describe several recent applications to problems of medical interest. After reviewing the molecular basis of the H/D exchange process, the different steps of the HXMS experiment--labeling, rapid proteolysis, fragment separation and mass measurement--are described, followed by a discussion of data analysis methods. Finally, we describe recent results on the application of HXMS to 1) mapping drug/inhibitor binding sites and detecting drug induced conformational changes, 2) studying viral capsid structure and assembly, and 3) characterizing the structure of pathological protein conformations, specifically amyloid fibrils.

Structure parameters of solvated silver(I) ions in eight neat solvents were determined by extended X-ray absorption fine structure spectroscopy. The coordination geometry of the solvated silver(I) ion is four-coordinate tetrahedral at the Ag−O bond distances of 239 pm in trimethylphosphate, 239 pm in N,N-dimethylformamide, 238 pm in 1,1,3,3-tetramethylurea, and 238 pm in dimethyl sulfoxide as oxygen-donating solvents, and at the Ag−N bond distances of 229 pm in acetonitrile, 230 pm in 2-methylpyridine, 229 pm in n-propylamine, and 231 pm in ethylenediamine as nitrogen-donating solvents. According to our present ab initio molecular orbital calculations concerning the structure of the silver(I) ion bound by n molecules of hydrogen cyanide (n = 1−6) and acetonitrile (n = 1−5) in the gas phase, the maximal stabilization for the solvation is observed at n = 4. The results of the theoretical calculations in the gas phase are consistent with the experimental observations in solution.