Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

How synonymous mutations alter enzyme structure and function over long timescales.

The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.

A proteome-wide map of chaperone-assisted protein refolding in a cytosol-like milieu.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients. Here, we develop a limited proteolysis-mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional.

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

Nonrefoldability is Pervasive Across the E. coli Proteome.

Decades of research on protein folding have primarily focused on a subset of small proteins that can reversibly refold from a denatured state. However, these studies have generally not been representative of the complexity of natural proteomes, which consist of many proteins with complex architectures and domain organizations. Here, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes using mass spectrometry-based proteomics. Our study covers the majority of the soluble E. coli proteome expressed during log-phase growth, and among this group, we find that one-third of the E. coli proteome is not intrinsically refoldable on physiological time scales, a cohort that is enriched with certain fold-types, domain organizations, and other biophysical features. We also identify several properties and fold-types that are correlated with slow refolding on the minute time scale. Hence, these results illuminate when exogenous factors and processes, such as chaperones or cotranslational folding, might be required for efficient protein folding.

A Structural Model of Nitro-Porphyrin Dyes Based on Spectroscopy and Density Functional Theory.

Nitro-porphyrins are an important class of commercial dyes with a range of potential applications. The nitro group is known to dramatically affect the photophysics of the porphyrin, but there are few systematic investigations of the contributing factors. To address this deficiency, we present spectroscopic studies of a series of nitro-porphyrins, accompanied by density functional theory calculations to elucidate their structures. In particular, we explore how the positions of the substituents affect the energy levels and nuclear geometry. As expected, nitro groups on the meso-phenyl rings cause small changes to the orbital energies by induction, while those at the ß-pyrrole positions more strongly conjugate into the aromatic system. In addition, however, we find evidence that ß-pyrrole nitro groups distort the porphyrin, creating two non-planar conformations with distinct properties. This unexpected result helps explain the anomalous photophysics of nitro-porphyrins reported throughout the literature, including inhomogeneous line broadening and biexponential fluorescence decay. © 2017 Wiley Periodicals, Inc.

The Accuracy of Common Hand Injections With and Without Ultrasound: An Anatomical Study.

BACKGROUND: The aim of the study is to determine the accuracy of hand injections with and without the aid of ultrasound (U/S) into the carpal tunnel, thumb carpometacarpal (CMC) joint, first dorsal compartment (DC) and the radiocarpal (RC) joint. METHODS: Four participants of various level of experience injected the carpal tunnel, thumb CMC, first DC, and RC joint into 40 fresh frozen cadaver specimens with blue dye and radiographic contrast. Participants 1 and 2 were injected without U/S guidance, and participants 3 and 4 were injected with U/S guidance. A successful injection was determined by both fluoroscopy and dissection/direct observation. Additional information was recorded for each injection such as median nerve infiltration and evidence of thumb CMC arthrosis. RESULTS: The overall accuracy for carpal tunnel, thumb CMC, first DC, and RC injections were 95%, 63%, 90%, and 90%, respectively. Success was compared with and without U/S guidance. Success rates were similar for each injection site, except the thumb CMC joint, where U/S participants had 25% higher accuracy. In the setting of thumb CMC arthrosis, the incidence of success was 38% for participants with no U/S aid and 72% for participants with U/S aid. There was a significant difference between participants who used U/S with the participant with more U/S experience being more successful. CONCLUSION: Carpal tunnel, first DC, and RC injections had an accuracy of greater than 90%. Thumb CMC injections have a lower accuracy (63%) and one can improve accuracy with U/S. The accuracy of U/S-guided injections is dependent on the user and their experience.

Retrospective Review of Air Transportation Use for Upper Extremity Amputations at a Level-1 Trauma Center.

INTRODUCTION: Air transportation to tertiary care centers of patients with upper extremity amputations has been utilized in hopes of reducing the time to potential replantation; however, this mode of transportation is expensive and not all patients will undergo replantation. The purpose of this study is to review the appropriateness and cost of air transportation in upper extremity amputations. MATERIALS AND METHODS: Consecutive patients transported by aircraft with upper extremity amputations in a 7-year period at a level-1 trauma center were retrospectively reviewed. The distance traveled was recorded, along with the times of the injury, referral, transportation duration, arrival, and start of the operation. The results of the transfer were defined as replantation or revision amputation. RESULTS: Overall, 47 patients were identified with 43 patients going to the operating room, but only 14 patients (30%) undergoing replantation. Patients arrived at the tertiary hand surgery center with a mean time of 182.3 minutes following the injury, which includes 105.2 minutes of transportation time. The average distance traveled was 105.4 miles (range, 22-353 miles). The time before surgery of those who underwent replantation was 154.6 minutes. The average cost of transportation was $20,482. DISCUSSION: Air transportation for isolated upper extremity amputations is costly and is not usually the determining factor for replantation. The type of injury and patients' expectations often dictate the outcome, and these may be better determined at the time of referral with use of telecommunication photos, discussion with a hand surgeon, and patient counseling. LEVEL OF EVIDENCE: III.

Character, Incidence, and Predictors of Knee Pain and Activity After Infrapatellar Intramedullary Nailing of an Isolated Tibia Fracture.

OBJECTIVE: To study the activity and incidence of knee pain after sustaining an isolated tibia fracture treated with an infrapatellar intramedullary nail at 1 year. DESIGN: Retrospective review of prospective cohort. SETTING: Multicenter Academic and Community hospitals. PATIENTS: Four hundred thirty-seven patients with an isolated tibia fracture completed a 12-month assessment on pain and self-reported activity. INTERVENTION: Infrapatellar intramedullary nail. OUTCOMES: Demographic information, comorbid conditions, injury characteristics, and surgical technique were recorded. Knee pain was defined on a 1-7 scale with 1 being "no pain" and 7 being a "very great deal of pain." Knee pain >4 was considered clinically significant. Patients reported if they were "able," "able with difficulty," or "unable" to perform the following activities: kneel, run, climb stairs, and walk prolonged. Variables were tested in multilevel multivariable regression analyses. RESULTS: In knee pain, 11% of patients reported a "good deal" to a "very great deal" of pain (>4), and 52% of patients reported "no" or "very little" pain at 12 months. In activity at 12 months, 26% and 29% of patients were unable to kneel or run, respectively, and 31% and 35% of patients, respectively, stated they were able with difficulty or unable to use stairs or walk. CONCLUSIONS: Clinically significant knee pain (>4/7) was present in 11% of patients 1 year after a tibia fracture. Of note, 31%-71% of patients had difficulty performing or were unable to perform routine daily activities of kneeling, running, and stair climbing, or walking prolonged distances. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

The most cited articles in hand surgery over the past 20-plus years: a modern-day reading list.

PURPOSE: To create a current reading list of the hand surgery articles most commonly cited in the last 20-plus years. METHODS: Using the Web of Science Citation Index Search, we searched "hand" and "wrist" in the orthopedic, surgery, and sport sciences research areas. We then reviewed the articles and chose the 50 most commonly cited articles related to hand surgery. Articles were categorized as clinical or basic science. Clinical articles were subcategorized as either therapeutic, prognostic, diagnostic, or economic/decision analysis and assigned a level of evidence rating. We calculated the number of citations per year (citation density). RESULTS: The total number of citations for the top 50 articles ranged from 92 to 317. Citation density ranged from 4 to 24 (average, 9.7). Of the 50 articles, 39 were clinical (78%), whereas the remainder were basic science. Clinical articles were most commonly therapeutic (25 of 39; 64%), followed by diagnostic (11 of 39; 28%) and prognostic (3 of 39; 8%). There were no economic/decision analysis-type articles. The most common level of evidence was level IV, which made up 38% of the list (19 of 50 articles). The second most common was level I, which represented 20% of the list (10 of 50 articles). A total of 70% of the articles (35 of 50) were published between 1990 and 1999, and the remainder of the articles were published after 1999. Fifty percent of the articles appeared in the Journal of Hand Surgery, American volume. CONCLUSIONS: Many of the articles found on our list have shaped the way we practice hand surgery today. We hope that this report and the articles it names can help residents and fellows study current hand surgery and its evolution over the past 20 years. TYPE OF STUDY/LEVEL OF EVIDENCE: Economic and decision analyses IV.

The palmar fat pad is a reliable intraoperative landmark during carpal tunnel release.

PURPOSE: During endoscopic or open carpal tunnel release, a palmar fat pad is visualized immediately proximal to the distal end of the transverse carpal ligament (TCL). Visualization of the fat pad allows anticipation of complete release of the TCL without unnecessary distal dissection that could risk iatrogenic injury. This study defines the anatomic relationship of the distal edge of the TCL to the fat pad, the superficial palmar arch, and the motor branch of the median nerve. METHODS: Eighteen fresh-frozen cadaver hands were dissected, and the proximal aspect of the palmar fat pad, the distal edge of the TCL, the superficial palmar arch, and the motor branch of the median nerve were identified. An electronic caliper was used to measure distances between each structure along the axis of the radial border of the ring finger. A subset of 8 hands was radiographically imaged with fingers flexed and extended (wrist neutral) to determine if finger positioning influenced measurements between marked structures. RESULTS: The proximal aspect of the palmar fat pad is 2.0 mm proximal to the distal edge of the TCL. The distal end of the TCL, as measured along the axis of the radial border of the ring finger is 12.7 mm from the most proximal aspect of the palmar arch and 6.5 mm from the nearest aspect of the motor branch. Flexing the fingers decreases the distance between the distal end of the TCL and the fat pad while not markedly affecting the distance between the TCL and the palmar arch or the motor branch. CONCLUSIONS: The palmar fat pad is a reliable anatomic landmark during carpal tunnel release. When dividing the TCL from proximal to distal, visualization of the proximal aspect of the fat pad indicates that the distal edge of the TCL is within approximately 2 mm and indicates that distal dissection beyond this extent is unnecessary.