Hydrogen exchange identifies native-state motional domains important in protein folding

Kim, Key Sun; Fuchs, James A.; Woodward, Clare

doi:10.1021/bi00088a012

Cited by 202 publications

(183 citation statements)

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“…Spectra were collected every 20 min for 16 h. The results showed that that all but six protons were exchanged within 20 min, the time required to acquire the first spectrum. The peaks that did persist were fully exchanged after 3 h. The exchange rate for the measurable peaks is consistent with the AG measured by urea denaturation (Kim et al, 1993). These results show that the majority of backbone NH groups of p16 are in very rapid exchange, therefore suggesting that p16 is a highly dynamic protein.…”

Section: Ja Boice and R Faimansupporting

confidence: 83%

“…We also measured protein dynamics using hydrogen-deuterium exchange rates as monitored by NMR. The rate at which amide protons exchange with solvent deuterons is an indication of the dynamics of a protein as well as the number of residues that are involved in the folding core (Kim et al, 1993). A sample of S-tagp16 was reconstituted in deuterated H,O and the NMR spectrum was compared to the NMR spectrum established for an identical sample reconstituted in H,O.…”

Section: Ja Boice and R Faimanmentioning

confidence: 99%

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Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Boice¹,

Fairman²

1996

Protein Science

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The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 pM. The CD spectrum indicates that the protein is composed of 33% a-helix, 22% P-sheet, 19% p-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a AG of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogendeuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.Keywords: analytical ultracentrifugation; ankyrin-like repeat; cyclin; circular dichroism; cyclin dependent kinase inhibitor Control of the eukaryotic cell cycle is a highly regulated process that involves the interaction of a large number of macromolecules. Critical checkpoints in this process are found during the entry of cells into both the GUS and G2/M phases of the cell cycle (for reviews, see Draetta, 1994;Sherr, 1995). The cyclin-dependent kinases (CDKs) are enzymes whose activities govern these checkpoints. CDK holoenzymes, which consist of a CDK catalytic subunit and a cyclin regulatory subunit, are regulated, in part, by their temporal expression, degradation, phosphorylation, and dephosphorylation. This regulation throughout the cell cycle serves to drive the cell through the successive phases.Recent studies have identified CDK inhibitors, a new class of proteins, adding yet another level of control to CDK regulation (for review, see Elledge & Harper, 1994). These CDK inhibitors are thought to function by blocking and/or disrupting CDK-cyclin association. p16, one of the first of these inhibitors to be discovered, was originally cloned from a yeast-two hybrid interaction screen using CDK4 as the bait protein (Serrano et al., 1993).

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Section: Ja Boice and R Faimansupporting

confidence: 83%

Section: Ja Boice and R Faimanmentioning

confidence: 99%

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Boice¹,

Fairman²

1996

Protein Science

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“…4b and formalized in reaction Scheme 1 (the infrared foldon was found later). This interesting possibility, that progressively slower exchanging sets of amide hydrogens might represent a sequential unfolding pathway, had been suggested much earlier on other grounds (Englander & Kallenbach, 1983;Kim et al 1993). …”

Section: Pufs To Pathwaysmentioning

confidence: 82%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

171

267

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Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

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“…It is particularly striking that at 5 "C, the N-terminal 310-helix and residues 54-56 of the C-terminal a-helix are more protected from exchange in Clarke et al, 1993;Kim et al, 1993;Woodward, 1993).…”

Section: Comparison Of Amide Proton Exchange Propertiesmentioning

confidence: 99%

Hydrogen exchange in BPTI variants that do not share a common disulfide bond

Schulman

Kim

1994

Protein Science

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Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-5 1, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-%]A], and [30-51; 14-383~5~55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution, However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI foldiing intermediates containing either the 5-55 or the 30-51 disulfide bond.

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Hydrogen exchange identifies native-state motional domains important in protein folding

Cited by 202 publications

References 75 publications

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

Protein folding and misfolding: mechanism and principles

Hydrogen exchange in BPTI variants that do not share a common disulfide bond

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