Abstract The rate of exchange peptide group NH hydrogens with the aqueous solvent is sensitive to neighboring side chains. To evaluate effects protein chains, all 20 naturally occurring amino acids were studied using dipeptide models. Both inductive and steric blocking are apparent. additivity nearest‐neighbor was tested in oligo‐and polypeptides and, suprisingly, confirmed. Reference rates for alanine‐containing peptides determined temperature considered. These results provide information...

The hydrogen exchange behavior of native cytochrome c in low concentrations denaturant reveals a sequence metastable, partially unfolded forms that occupy free energy levels reaching up to the fully state. step from one form another is accomplished by unfolding or more cooperative units structure. are entire omega loops mutually stabilizing pairs whole helices and loops. detected appear represent major intermediates reversible, dynamic reactions occur even at conditions thus may define...

Abstract The hydrogen exchange (HX) rates of the slowest peptide group NH hydrogens in oxidized cytochrome c (equine) are controlled by transient global unfolding equilibrium. These can be measured one‐dimensional nuclear magnetic resonance and used to determine thermodynamic parameters at mild solution conditions well below melting transition. free energy for differ from values found standard denaturation methods, most notably due slow cis‐trans isomerization prolyl bond. This difference...

Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units continually unfold refold even under native conditions. Folding proceeds by the stepwise assembly rather than one amino acid at a time. The folding pathway determined sequential stabilization process; previously formed foldons guide stabilize subsequent to progressively build protein. Four other proteins have been found similar behavior. These results support protein pathways through...

Experiments with cytochrome c (cyt c) show that an initial folding event, molecular collapse, is not energetically downhill continuum as commonly presumed but represents a large-scale, time-consuming, cooperative barrier-crossing process. In the absence of later misfold-reorganization barriers, early collapse barrier limits cyt to time scale milliseconds. The process itself appears be limited by uphill search for some coarsely determined transition state structure can nucleate subsequent...

The structure of α-synuclein (α-syn) amyloid was studied by hydrogen-deuterium exchange using a fragment separation–MS analysis. conditions used made it possible to distinguish the unprotected and protected amide hydrogens define order/disorder boundaries at close amino acid resolution. soluble α-syn monomer exchanges its with water random coil rates, consistent natively unstructured condition. In assembled amyloid, long N-terminal C-terminal segments remain (residues 1–≈38 102–140),...

The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis an advanced fragment separation mass spectrometry technology. results show that proceeds through distinct intermediates in a stepwise pathway sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as concerted transition one or more segments from HX-unprotected HX-protected state. Deconvolution data near amino acid resolution...

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology study large biologically important proteins. A persistent problem is degradation information due back deuterium label during fragmentation-separation process needed prepare samples for MS measurement. This paper reports a systematic factors that influence (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates,...

Apolipoprotein A-I (apoA-I) stabilizes anti-atherogenic high density lipoprotein particles (HDL) in the circulation and governs their biogenesis, metabolism, functional interactions. To decipher these important structure-function relationships, it will be necessary to understand structure, stability, plasticity of apoA-I molecule. Biophysical studies show that lipid-free contains a large amount alpha-helical structure but location this its properties are not established. We used...

Significance This paper shows how hydrogen exchange–mass spectrometry data can be deconvolved to obtain direct protein structural information at amino acid resolution. The solution this problem has eluded prior efforts and is considered of fundamental importance for the rapidly expanding exchange–MS field.

Significance This paper considers the experimental evidence for and against two major current models protein folding, theoretically hypothesized many-pathway model, experiment-based defined-pathway model. The questions of how proteins fold, why they fold in that way, folding pathway each is encoded its sequence structure have fundamental significance design, misfolding, regulation function, clinical problems, industrial applications. present analysis attempts to distinguish answer these questions.

Measurement of the naturally occurring hydrogen exchange (HX) behavior proteins can in principle provide highly resolved thermodynamic and kinetic information on protein structure, dynamics, interactions. The HX fragment separation-mass spectrometry method (HX-MS) is able to measure biologically important systems that are not accessible NMR methods. In order achieve high structural resolution HX-MS experiments, it will be necessary obtain many sequentially overlapping peptide fragments...

A previous paper considered the problems that presently limit hydrogen exchange-mass spectrometry (HX-MS) method for studying biophysical and functional properties of proteins. Many these can be overcome by obtaining analyzing hundreds sequentially overlapping peptide fragments cover protein many times over (Mayne et al. J. Am. Soc. Mass Spectrom. 2011: 10.1007/s13361-011-0235-4). This describes a computer program called ExMS furthers this advance making it possible to efficiently process...

The analysis of many hydrogen exchange (HX) experiments depends on knowledge rates expected for the unstructured protein under same conditions. We present here some minor adjustments to previously calibrated values and a stringent test their accuracy. Graphical Abstract ᅟ.

Abstract The exchange of a large number amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to through local fluctuations larger unfolding reactions were separately considered. All protected from factors greater than 10 3 are defined H‐bonds, almost all H‐bonded including those at the protein surface slowly. H‐exchange rates do not correlate H‐bond strength (length) or crystallographic B factors. It appears that...

To test the significance of ultrafast protein folding signals (≪1 msec), we studied cytochrome c (Cyt c) and two Cyt fragments with major C-terminal segments deleted. The remain unfolded under all conditions so could be used to define baselines for fluorescence circular dichroism (CD) as a function denaturant concentration. When diluted from high low in kinetic experiments, readjust their new baseline values “burst phase” within mixing dead time. fragment burst phase reflects contraction...